Methods for detecting the presence, location or quantity of targets using novel dyes

ABSTRACT

The present invention provides dyes, reactive dyes and labeled reagents that may be used in the detection or quantification of desirable target molecules, such as proteins and nucleic acids. Dyes are provided that may be used free in solution where the binding of the dye to the target molecule provides signal generation. Dyes are also provided that comprise reactive groups that may be used to attach the dyes to probes that will bind to desirable target molecules. The novel dyes of the present invention have been modified by the addition of charged and polar groups to provide beneficial properties.

RELATED APPLICATION DATA

This application is a divisional of U.S. patent application Ser. No.11/137,771, filed on May 24, 2005, now allowed, the contents of whichare incorporated herein by reference. This application claims priorityto the aforementioned Ser. No. 11/137,771, filed on May 24, 2005.

FIELD OF THE INVENTION

This invention relates to field of labeling compositions, reagents andprocesses that are useful in applications related to the detection,quantification and localization of target molecules of interest thatinclude nucleic acids and proteins.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

There are a variety of properties that might be desirable for dyes thatare intended for use as markers for detection of proteins or nucleicacid hybridization. These can include the ability to bind to a protein,lipid or nucleic acid, the capability of incorporation into nucleicacids by enzymatic means when attached to a nucleotide, a lack of sterichindrance that could potentially interfere with hybridization, watersolubility, lack of aggregation, ability to intercalate intodouble-stranded nucleic acids and the presence of a reactive group thatallows attachment of the dye to a nucleotide or other desirable target.Suitable dyes could have many of these properties but do not need tohave them all. For instance, the ability to intercalate may allowdetection of hybridization events in the presence of unhybridized probesor it may provide increased hybridization stabilization. Examples ofthese applications are disclosed in European Patent Application EP 0 231495, U.S. Pat. No. 5,994,056 and U.S. Pat. No. 6,174,670, all of whichare incorporated by reference. Similarly, the ability to be incorporatedby an enzyme is a useful property when carrying out enzymatic labelingof nucleic acids. Labels that are inhibitory towards incorporation canstill be used in some methods where nucleic acids are chemicallysynthesized rather than using enzymatic means. Also, nucleotides withreactive groups such as allyl-amine may be incorporated enzymaticallyinto nucleic acids and then in a second step they are post-syntheticallymodified by attachment of dyes. Steric hindrance may be compensated tosome degree by the nature of the linker joining the dye to a nucleotidewith regard to both the length and the constituents of the linker. For adiscussion of this last point, see U.S. Patent Application Ser. No.2003/0225247, hereby incorporated by reference.

The particular spectral characteristics of dyes are also importantqualities. Although broad-spectrum white light can be used as a sourceof excitation, lasers with defined set wavelengths are most commonlyemployed. As such, dyes that would find most immediate use would haveexcitation wavelengths that can make use of such laser emissions.Emission wavelengths are of a more flexible nature since filters can beused to isolate a particular part of the spectrum. However, it should benoted that there are a number of machines used for detection of labelednucleic acids that have been designed with dyes that are commonly used.For instance, there are a number of slide scanners that have beenoptimized for detection of nucleic acids labeled with the Cy3 and Cy5dyes described by Waggoner et al. in U.S. Pat. No. 5,268,486(incorporated herein by reference). On the other hand, the availabilityof dyes that have useful properties but have wavelengths that are notcommonly used can prove to be an incentive to adopt lasers withcompatible wavelengths.

A set of dyes with well separated emission spectra may find use wheremore than one fluorophor is to be used at the same time. Well knownapplications in this are immunostaining for various proteins in cells,in situ hybridization for multiple targets, non-radioactive sequencing,nucleic acid array analysis, protein array analysis, as well asnon-specific cellular staining with dyes having general affinities forproteins or lipids. On the other hand, overlapping spectralcharacteristics also have applications; for instance, emission by onefluorophor may be used to excite a second fluorophor through energytransfer when distances are sufficiently close.

Among the dyes that have been most widely used as markers for proteinsand nucleic acid labeling are members of the xanthene, coumarin, cyanineand asymmetric cyanine dye families. Xanthene dyes are among theearliest dyes used for biological staining, where fluorescein was usedto work out many of the techniques for labeling proteins and nucleicacids. The basic structure of fluorescein molecules can be depicted as:

Related xanthene compounds that have also been used as labels includerhodols and rhodamines. Their basic structure is as follows:

The R group attached to the central structure is typically a substitutedphenyl group although as described in U.S. Patent Application Ser. No.2003/0225247 (hereby incorporated by reference), aphenylic versions arealso suitable as dyes.

Another family of dyes that have enjoyed widespread use is based uponderivatives of coumarin. The basic structure of coumarin is as follows:

Typically, coumarin derivatives will be dyes when R is an OH or an aminegroup. Useful compounds have also been made where R is further modifiedsuch that an enzymatic cleavage event converts the R group into an OH oramine group. Thus this proto-dye or dye precursor can be used as markerfor the presence of an enzyme that is capable of converting a coumarincompound into a fluorescent dye. Discussions of such methods aredisclosed in U.S. Pat. No. 5,696,157 and U.S. Pat. No. 5,830,912, bothof which are incorporated by reference.

As described above, a large number of useful dyes are based upon cyaninedyes. The basic structure of Cyanine dyes is as follows

As will be discussed later, major factors in the particular spectralqualities of these dyes is dependent upon the number “n”, the nature of“X” and “Y” and functional groups that extend the aromaticity of thedyes.

Other compounds that were functionally considered to be Cyanine-typedyes (see U.S. Pat. No. 5,268,486 hereby incorporated by reference) arethe merocyanine and styryl dyes whose structures are:

There are a variety of atoms that have been used in the X and Ypositions. These have included carbon, sulfur, oxygen, nitrogen andselenium. When X or Y is a carbon, this portion of the dye is anindolinium moiety. When X or Y is substituted by sulfur, oxygen ornitrogen this portion is respectively described as a benzothiazolium,benzoxazolium or a benzimidazolium moiety.

Another version of styryl dyes can have picoline or quinoline moietiesinstead of the benzazolium group, thereby having the structures:

Asymmetric cyanine dyes contain one portion that is essentially thebenzazolium portion of the cyanine dye family but connected to thisportion by the methine bridge is a different aromatic compound. Theirstructure is as follows:

The aromatic moiety can be a six membered aromatic or heteroaromaticring

Improvements to these dyes have been carried out by substitution ofvarious groups onto the basic structure, i.e. on the carbons andnitrogens of the preceding structures or where H or R groups arefeatured. Additionally, other rings may be fused to various parts of therings in the structures above, thereby generating more complexstructures. These modifications have led to shifts in the excitation andemission characteristics of the dyes that allow a large number of dyeswith same basic structure but having different spectral characteristics,i.e. modifications can be made in their structure that can alter theparticular wavelengths where these compounds will absorb and fluorescelight. As described above, the cyanine dyes can have a general structurecomprising two benzazolium-based rings connected by a series ofconjugated double bonds. The dyes are classified by the number (n) ofcentral double bonds connecting the two ring structures;monocarbocyanine or trimethinecarbocyanine when n=1; dicarbocyanine orpentamethinecarbocyanine when n=2; and tricarbocyanine orheptamethinecarbocyanine when n=3. The spectral characteristics of thecyanine dyes have been observed to follow specific empirical rules. Forexample, each additional conjugated double bond between the ringsusually raise the absorption and emission maximum about 100 nm. Thus,when a compound with n=1 has a maximum absorption of approximately 550nm, equivalent compounds with n=2 and n=3 can have maximum absorptionsof 650 nm and 750 nm respectively. Addition of aromatic groups to thesides of the molecules has lesser effects and may shift the absorptionby 15 nm to a longer wavelength. The groups comprising the indoleninering can also contribute to the absorption and emission characteristics.Using the values obtained with gem-dimethyl group as a reference point,oxygen substituted in the ring for the gem-dimethyl group can decreasethe absorption and emission maxima by approximately 50 nm. In contrast,substitution of sulfur can increase the absorption and emission maximaby about 25 nm. R groups on the aromatic rings such as alkyl,alkyl-sulfonate and alkyl-carboxylate usually have little effect on theabsorption and emission maxima of the cyanine dyes (U.S. Pat. No.6,110,630, hereby incorporated by reference).

As described above, alteration of spectral qualities is only one usefulmodification that can be made to a dye. In another instance,modification of a dye by a sulfonate group may increase the stability ofmany dyes and thereby resist “bleaching” after illumination.Modification of dyes by sulfonation was later applied in themodification of cyanine dyes with reactive groups (U.S. Pat. No.5,569,766 hereby incorporated by reference), where it was reported thatthe sulfonation decreases aggregation of labeled materials. It wasfurther applied to xanthenes, coumarins and the non-benzazolium portionof asymmetric cyanine dyes (U.S. Pat. No. 5,436,134, U.S. Pat. No.6,130,101 and U.S. Pat. No. 5,696,157, all of which are herebyincorporated by reference). Modifications of dyes haves also been madeto increase their affinity or selectivity towards binding to nucleicacids (European Patent Application Serial No. EP0 231495, U.S. PatentApplication Serial No. 2003/0225247 and U.S. Pat. No. 5,658,751, all ofwhich are incorporated by reference).

In many cases, the utility of these dyes has been achieved by synthesisof compounds with a reactive group that allows attachment of the dye toa target molecule. For instance, although cyanine dyes in themselves hadbeen known for many years, it was only when derivatives were describedwith reactive groups (U.S. Pat. No. 5,268,486 hereby incorporated byreference) that they found widespread use in labeling proteins andnucleic acids. Their versatility was then increased by disclosure ofother groups that could be used to attach cyanine dyes to suitablepartners (U.S. Pat. No. 6,114,350 and U.S. Patent Application Ser. No.2003/0225247, both of which are hereby incorporated by reference). Anexemplarary list of electrophilic groups and corresponding nucleophilicgroups that can be used for these purposes are given in Table 1 of USPat. No. 6,348,596 (hereby incorporated by reference).

A variety of linker arms may be used to attach dyes to targets. Commonlyused constituents for linkers are chains that contain varying amounts ofcarbon, nitrogen, oxygen and sulfur. Examples of linkers using some ofthese combinations are given in U.S. Pat. No. 4,707,440, herebyincorporated by reference. Bonds joining together the constituents canbe simple carbon-carbon bonds or they may be acyl bonds (U.S. Pat. No.5,047,519), sulfonamide moieties (U.S. Pat. No. 6,448,008) and polargroups (U.S. Patent Application Ser. No. 2003/0225247) all of which arehereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention provides a dye having the formula:

wherein X comprises CR¹¹R¹², NR¹¹, O, S or Se where R¹¹ and R¹²independently comprise hydrogen, a halogen, an amino group, or an alkylgroup. The alkyl group is saturated or unsaturated, linear or branched,substituted or unsubstituted, an alkoxy group wherein the alkoxy groupis saturated or unsaturated, branched or linear, substituted orunsubstituted, or when taken together, R¹¹ and R¹² form a five- orsix-membered ring. In the formula given above, n can be 0, 1, 2 or 3;wherein Y is —CR⁹═CR¹⁰. In the formula, m and p can have values of 0 or1 and m+p=1; wherein at least one of R¹, R², R³, R⁴, R⁵, R¹¹ or R¹²comprises Q(1) or at least one of R⁶, R⁷, R⁸, R⁹ or R¹⁰ comprises Q(2).Q(1) comprises a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), asulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide(SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), aphosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonatemonoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S. Q(1) is attached directly, or indirectly through alinker arm comprising carbon, sulfur, oxygen, nitrogen, or anycombinations thereof, wherein the linker arm may be saturated orunsaturated, linear or branched, substituted or unsubstituted, or anycombinations thereof and wherein when Q(1) is a sulfonamide, it does notcomprise a terminal reactive group or a linker joining the dye to atarget molecule. Q(2) comprises a sulfoxide (SOR¹³), a sulfone(SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphonate (PO₃ ⁼), aphosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴), wherein any of E can independentlycomprise O or S. In this dye, Q(2) is attached directly, or indirectlythrough a linker arm comprising carbon, sulfur, oxygen, nitrogen, or anycombinations thereof, where the linker arm may be saturated orunsaturated, linear or branched, substituted or unsubstituted or anycombinations thereof. In this dye, when Q(2) is a sulfonamide, it doesnot comprise a terminal reactive group or a linker joining the dye to atarget molecule. In this dye, R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰ can behydrogen, a halogen, an amino group, an alkyl group wherein said alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group wherein the alkoxy group is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together R¹³ and R¹⁴ form a five- or six-membered ring. In thisdye, R¹⁶, R¹⁷, R¹⁸ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹ and R¹² can independently be hydrogen, Z, an alkyl groupwherein the alkyl group is saturated or unsaturated, linear or branched,substituted or unsubstituted, an alkoxy group that is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together, R² and R³, R³ and R⁴, R⁴ and R⁵, R⁶ and R⁷, and R⁷ andR⁸ may form a five- or six-membered ring; or when taken together R¹ andR¹⁶, R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁷ and R¹⁸, and R¹⁸ and R⁹ may form afive- or six-membered ring. In this dye Z comprises a carboxyl group(CO₂ ⁻), a carbonate ester (COER¹³), a sulfonate (SO₃ ⁻), a sulfonateester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), asulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester(PO₃ ⁻ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) aphosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴). In the foregoing, E can beindependently O or S; wherein Z is attached directly, or indirectlythrough a linker arm comprising carbon, sulfur, oxygen, nitrogen, or anycombinations thereof and wherein said linker arm may be saturated orunsaturated, linear or branched, substituted or unsubstituted or anycombinations thereof. Any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰,R¹¹ or R¹² may further comprise a heteroatom containing side chainwherein the side chain is joined to the R group by a linkage whichcomprises an ether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or anamine linkage (—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷independently comprise hydrogen, Z, an alkyl group wherein the alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five- orsix-membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise the heteroatom containing side chain.

The present invention also provides a dye having the formula:

wherein X comprises CR¹¹R¹², NR¹¹, O, S or Se where R¹¹ and R¹²independently comprise hydrogen, a halogen, an amino group, or an alkylgroup. The alkyl group is saturated or unsaturated, linear or branched,substituted or unsubstituted, an alkoxy group wherein the alkoxy groupis saturated or unsaturated, branched or linear, substituted orunsubstituted, or when taken together, R¹¹ and R¹² form a five- orsix-membered ring. In the formula above, n can be 0, 1, 2 or 3; Y is—CR⁹═CR¹⁰—; m and p can have values of 0 or 1 and m+p=1; and wherein atleast one of R¹, R², R³, R⁴, R⁵, R¹¹ or R¹² comprises Q(1) or at leastone of R⁶, R⁷, R⁸, R⁹ or R¹⁰ comprises Q(2). Q(1) comprises a sulfonate(SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone(SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), aphosphate monoester (PO₃ ^(−ER) ¹³), a phosphate diester (PO₂ER¹³ER¹⁴),a phosphonate (PO₃ ⁼) a phosphonate monoester (PO₂ ⁻ER¹³) a phosphonatediester (POER¹³ER¹⁴), a thiophosphate (PSO₃ ⁼), a thiophosphatemonoester (PSO₂ ⁻ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), athiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO⁻ER¹³) athiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)wherein any of E can independently comprise O or S. In this dye, Q(1) isattached directly, or indirectly through a linker arm comprising carbon,sulfur, oxygen, nitrogen, or any combinations thereof, wherein thelinker arm may be saturated or unsaturated, linear or branched,substituted or unsubstituted, or any combinations thereof and whereinwhen Q(1) is a sulfonamide, it does not comprise a terminal reactivegroup or a linker joining the dye to a target molecule. In the dye ofthe present invention Q(2) comprises a sulfoxide (SOR¹³), a sulfone(SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphonate (PO₃ ⁼), aphosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S. In this dye Q(2) is attached directly, or indirectlythrough a linker arm comprising carbon, sulfur, oxygen, nitrogen, or anycombinations thereof, wherein the linker arm may be saturated orunsaturated, linear or branched, substituted or unsubstituted or anycombinations thereof and wherein when Q(2) is a sulfonamide, it does notcomprise a terminal reactive group or a linker joining the dye to atarget molecule. In the foregoing formulae, R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰can be hydrogen, a halogen, an amino group or an alkyl group. The alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group wherein said alkoxy group is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together R¹³ and R¹⁴ form a five- or six-membered ring. In thisdye, R¹⁶, R¹⁷, R¹⁸ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹ and R¹² can independently be hydrogen, Z, or an alkyl groupwherein the alkyl group is saturated or unsaturated, linear or branched,substituted or unsubstituted, an alkoxy group that is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together, R² and R³, R³ and R⁴, R⁴ and R⁵, R⁶ and R⁷, and R⁷ andR⁸ may form a five- or six-membered ring; or when taken together R¹ andR¹⁶, R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁷ and R¹⁸, and R¹⁸ and R⁹ may form afive- or six-membered ring. In the dye Z comprises a carboxyl group (CO₂⁻), a carbonate ester (COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester(SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide(SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), aphosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonatemonoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) where E can be independently O or Sand the structures are as described previously. As just provided Z isattached directly, or indirectly through a linker arm comprising carbon,sulfur, oxygen, nitrogen, or any combinations thereof, wherein thelinker arm may be saturated or unsaturated, linear or branched,substituted or unsubstituted or any combinations thereof.

Any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰, R¹¹ or R¹² may furthercomprise a heteroatom containing side chain, wherein the side chain isjoined to the R group by a linkage which comprises an ether linkage(—OR²⁵), a thioether linkage (—SR²⁵), or an amine linkage (—NR²⁵R²⁶ or—N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independently comprisehydrogen, Z, an alkyl group wherein the alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup that is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R²⁵ and R²⁶, and R²⁶ and R²⁷independently comprise a five- or six-membered ring, and wherein any ofR²⁵, R²⁶ or R²⁷ may further comprise the heteroatom containing sidechain. In this dye, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹³, R¹⁴, or R¹⁵ further comprises a nucleophilic reactive group,an electrophilic reactive group, a terminal alkene, a terminal alkyne, acoordinate group or an alkylating agent.

Also provided by the present invention is a composition comprising afirst portion and a second portion, wherein the first portion comprisesa dye and the second portion comprises a target molecule, the dye havingthe formula:

wherein X comprises CR¹¹R¹², NR¹¹, O, S or Se where R¹¹ and R¹²independently comprise hydrogen, a halogen, an amino group, an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein thealkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken together, R¹¹ and R¹² form afive- or six-membered ring. In this composition and the formula above, ncan be 0, 1, 2 or 3; Y is —CR⁹═CR¹⁰—; m and p can have values of 0 or 1and m+p=1; and wherein at least one of R¹, R², R³, R⁴, R⁵, R¹¹ or R¹²comprises Q(1) or at least one of R⁶, R⁷, R⁸, R⁹ or R¹⁰ comprises Q(2).In this composition Q(1) comprises a sulfonate (SO₃ ⁻), a sulfonateester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), asulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester(PO₃ ⁻ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) aphosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ^(−ER) ¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S. In this composition Q(1) is attached directly, orindirectly through a linker arm comprising carbon, sulfur, oxygen,nitrogen, or any combinations thereof and wherein the linker arm may besaturated or unsaturated, linear or branched, substituted orunsubstituted, or any combinations thereof and wherein when Q(1) is asulfonamide, it does not comprise a terminal reactive group or a linkerjoining the dye to a target molecule. Q(2) comprises a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphonate (PO₃ ⁼), a phosphonate monoester (PO₂ ⁻ER¹³) a phosphonatediester (POER¹³ER¹⁴), a thiophosphate (PSO₃ ⁼), a thiophosphatemonoester (PSO₂ ⁻ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), athiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO⁻ER¹³) athiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)wherein any of E can independently comprise O or S. In this compositionQ(2) is attached directly, or indirectly through a linker arm comprisingcarbon, sulfur, oxygen, nitrogen, or any combinations thereof andwherein the linker arm may be saturated or unsaturated, linear orbranched, substituted or unsubstituted or any combinations thereof andwherein when Q(2) is a sulfonamide, it does not comprise a terminalreactive group or a linker joining the dye to a target molecule. In theforegoing formulae R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰ can be hydrogen, ahalogen, an amino group, an alkyl group wherein the alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group wherein the alkoxy group is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together R¹³ and R¹⁴ form a five- or six-membered ring. R¹⁶, R¹⁷,R¹⁸ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ andR¹² can independently be hydrogen, Z, an alkyl group wherein the alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R² and R³, R³ and R⁴, R⁴ and R⁵, R⁶ and R⁷, and R⁷ and R⁸ mayform a five- or six-membered ring; or when taken together R¹ and R¹⁶,R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁷ and R¹⁸, and R¹⁸ and R⁹ may form a five-or six-membered ring. In this composition Z comprises a carboxyl group(CO₂ ⁻), a carbonate ester (COER¹³), a sulfonate (SO₃ ⁻), a sulfonateester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), asulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester(PO₃ ⁻ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) aphosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) where E can be independently O or Sand the structures are as described previously. In this composition Z isattached directly, or indirectly through a linker arm comprising carbon,sulfur, oxygen, nitrogen, or any combinations thereof and wherein thelinker arm may be saturated or unsaturated, linear or branched,substituted or unsubstituted or any combinations thereof. In thiscomposition.

Any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰, R¹¹ or R¹² may furthercomprise a heteroatom containing side chain wherein the side chain isjoined to the R group by a linkage which comprises an ether linkage(—OR²⁵), a thioether linkage (—SR²⁵), or an amine linkage (—NR²⁵R²⁶ or—N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independently comprisehydrogen, Z, an alkyl group wherein the alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup that is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R²⁵ and R²⁶, and R²⁶ and R²⁷independently comprise a five- or six-membered ring, and wherein any ofR²⁵, R²⁶ or R²⁷ may further comprise the heteroatom containing side. Atleast one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹³, R¹⁴, or R¹⁵is linked to said second portion.

The present invention further provides a method for detecting thepresence or quantity of a target comprising the steps of: a) providingi) a sample where the presence or quantity of a target is desired to bedetected ii) a composition comprising a first portion and a secondportion wherein the first portion comprises a dye and the second portioncomprises a target specific moiety, the dye having the formula

as described and defined above; b) allowing any targets present in thesample i) to bind with the target specific moiety comprising thecomposition ii); and c) quantifying the amount of the composition ii)bound to any of the target in the sample, thereby detecting the presenceor quantity of the target.

Also provided by the invention herein is a method for detecting thepresence, location or quantity of a target comprising the steps of: a)providing i) a sample where the presence, location or quantity of atarget is desired to be detected, ii) a dye, having the formula:

as described and defined above; b) allowing any targets present in thesample i) to bind with the dye ii); and c) detecting the dye ii) boundto any of the target in the sample i), thereby detecting the presence,location or quantity of the target.

Other processes are provided by the present invention including aprocess for increasing the amount of amplification of a target sequence,the process comprising amplifying the target sequence in the presence ofan intercalating compound at a concentration where the efficiency ofamplification is increased compared to the efficiency of amplificationin the absence of the intercalating compound.

Another process provided by the present invention is for increasing thespecificity of amplification of a target sequence, the processcomprising amplifying the target sequence in the presence of anintercalating compound at a concentration where the specificity ofamplification is increased compared to the specificity of amplificationin the absence of the intercalating compound.

Another process of the present invention is for increasing the amount ofamplification of a target sequence and for increasing the specificity ofamplification of a target sequence, the process comprising amplifyingthe target sequence in the presence of an intercalating compound at aconcentration where (i) the efficiency of amplification is increasedcompared to the efficiency of the intercalating compound, and (ii) thespecificity of amplification is increased compared to the specificity ofamplification in the absence of the intercalating compound.

Yet another process by the invention herein is for decreasing the amountof non-target amplification during a target amplification reaction, theprocess comprising amplifying the target sequence in the presence of anintercalating compound at a concentration where the efficiency ofamplification of a non-target sequence is decreased compared to theefficiency of amplification of a non-target sequence in the absence ofthe intercalating compound.

DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph of samples from Example 33 assayed by gelelectrophoresis.

FIG. 2 is a photograph of samples from Example 34 assayed by gelelectrophoresis.

FIG. 3 is a photograph of samples from Example 35 assayed by gelelectrophoresis.

FIG. 4 is a photograph of samples from Example 36 assayed by gelelectrophoresis.

FIG. 5 is a photograph of samples from Example 37 assayed by gelelectrophoresis.

DESCRIPTION OF THE INVENTION

The present invention provides dyes, reactive dyes and labeled reagentsthat may be used in the detection or quantification of desirable targetmolecules. Some of these dyes may be used free in solution where thebinding of the dye to the target molecule provides increasefluorescence. Other dyes of the present invention comprise reactivegroups that may be used to attach the dyes to desirable targetmolecules. The novel dyes of the present invention have been modified bythe addition of charged groups as exemplified by sulfonates, phosphates,phosphonates and their derivatives. Other dyes have been modified by theaddition of polar groups such as sulfoxide, sulfone and sulfonamidemoieties. Dyes may also be modified by both charged and polar groups.

In the present invention, sulfonates are considered to be any group withthe formula SO₃ ⁻ including both sulfonic acid as well as varioussulfonate salts. The addition of a sulfonate group provides a chargedmoiety that can increase solubility, inhibit bleaching and reduceaggregation. The addition of phosphonate (PO₃ ⁼), phosphate (O—PO₃ ⁼)moieties or their derivatives may also provide such qualities.Transformation of the foregoing charged species into esters may converta charged group into a polar group. Derivatives that may find use withthe present invention can include thioanalogues such as thiophosphates,thiophosphonates and thioesters. Other derivatives that may find use caninclude phosphoramides and phosphonamides.

In the present invention, sulfones are considered to be any groups thathave the formula C—SO₂—C where carbon atoms are attached to theintervening sulfur atom. One of the carbon atoms may be part of a ringstructure of the dye or it may part of an intervening alkyl groupconnecting the sulfone to the dye. When one of the carbons of a sulfoneis replaced by a nitrogen atom the group is a sulfonamide.

The presence of the polar groups may help nucleotide incorporation sincedyes with polar groups will be less negatively charged than theirionized equivalents and thus be less repelled by the negatively chargedphosphate backbone of a nucleic acid template. The sulfone orsulfonamide group can be modified as desired by linkage to othermoieties to add desirable properties. It is also understood that thedegree of charge or polarity can be determined by the user by theaddition of appropriate combinations of charged and polar groups to adye.

In the present invention, Sulfoxides (SOR¹³), Sulfones (SO₂CR¹³R¹⁴R¹⁵)and sufonamides sulfonamides (SO₂NR¹³R¹⁴) are respectively defined ashaving the structures:

In the present invention, phosphates (PO₄ ⁼), their monoesters (PO₃⁻E⁻R¹³), diesters (PO₂ER¹³ER¹⁴), are respectively defined as having thestructures:

when E is an oxygen in the monoester and diester and

when E is a sulfur.

In the present invention, phosphonates (PO₃ ⁼), their esters (PO₂ ⁻ER¹³and POER¹³ER¹⁴) are respectively defined as having the structures:

when E is an oxygen in the monoester and diester and

when E is a sulfur.

In the present invention, thiophosphates (PSO₃ ⁼), their esters (PSO₂⁻ER¹³ and PSOER¹³ER¹⁴) are respectively defined as having thestructures:

when E is an oxygen in the monoester and diester and

when E is a sulfur.

In the present invention, thiophosphonates (PSO₂ ⁼), their esters(PSO⁻ER¹³ and PSER¹³ER¹⁴) are respectively defined as having thestructures:

when E is an oxygen in the monoester and diester and

when E is a sulfur

In the present invention, sulfonates (SO₃ ⁻), their esters (SO₂ER¹³) arerespectively defined as having the structures:

when E is an oxygen or sulfur in the ester linkage.

In the present invention, phosphonamides (PONR¹³R¹⁴NR¹⁹R²⁰),phosphoramides (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰) and phosphoramidites(PO₂R¹⁹NR¹³R¹⁴) are respectively defined as having the structures:

and their thioanalogues (PSNR¹³R¹⁴NR¹⁹R²⁰) (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰) and(POSR¹⁹NR¹³R¹⁴) having respectively the structures:

It is also understood that when a dye comprises anionic group, therewill also be a cationic counterion present. Any cation may serve thispurpose as long as it doesn't interfere with the use of the dye.Examples of cations that may serve as counterions can include but not belimited to hydrogen, sodium, potassium, lithium, calcium, cesium,ammonium, alkyl ammonium, alkoxy ammonium and pyridinium. It is alsounderstood that when a dye comprises a cationic group, there will alsobe an anionic counterion present. Any anion may serve this purpose aslong as it doesn't interfere with the use of the dye. Examples of anionsthat may serve as counterions can include but not be limited to halidessuch as a bromide, chloride, fluoride and iodide. Other examples caninclude but not be limited to perchlorate (ClO₄ ⁻), sulfate (SO₄ ⁼),sulfonate, alkane sulfonate, aryl sulfonate, phosphate, tosylate,mesylate and tetrafluoroborate moieties. In some cases the counterion orcounterions are provided by the dye being a salt where they exist asseparate ionic species. In other cases, the counterion or counterionsmay be present as part of the compound (sometimes called inner salts).It is understood that there may also be a combination of ions that areprovided by the compound and salts. With regard to acid moieties thatare shown in forms such as COOH it is also understood that thesecompounds may be found in ionized forms such as COO⁻.

Alkyl or alkoxy R groups may be substituted or unsubstituted. Examplesof substitutions can include but not be limited to one or more fluorine,chlorine, bromine, iodine, hydroxy, carboxy, carbonyl, amino, cyano,nitro or azido groups as well as other alkyl or alkoxy groups. Thelength of the alkoxy groups may be as desired. For instance, they mayindependently comprise from 1 to 18 carbons in length. They may beshorter as well, for instance they may be only 1 to 6 carbons in lengthin a dye molecule of the present invention.

The polar groups, charged groups and other substituents may be connectedto the dye directly or they may be connected by a linker arm comprisingcarbon, nitrogen, sulfur, oxygen or any combination thereof. The linkerarm may be saturated or unsaturated, linear or branched, substituted orunsubstituted as well as any combination of the foregoing.

In one aspect of the present invention, novel dyes that are based uponcyanine dyes are disclosed. In one embodiment the dyes have thestructure:

wherein X and Y independently comprise CR¹¹R¹², NR¹¹, O, S or Se,wherein R¹¹ and R¹² independently comprise hydrogen, a halogen, an aminogroup, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup wherein said alkoxy group is saturated or unsaturated, branched orlinear, substituted or unsubstituted, or when taken together, R¹¹ andR¹² comprise a 5 or 6 membered ring;

wherein n can be 0, 1, 2 or 3

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, orR¹² comprises Q, wherein Q comprises a sulfoxide (SOR¹³), a sulfone(SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphate monoester (PO₃⁻ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate monoester (PO₂⁻ER¹³), a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃ ⁼), athiophosphate monoester (PSO₂ ⁻ER¹³), a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³), a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴), or its thioanalogue (POSR¹⁹NR¹³R¹⁴),wherein any of E independently comprises O or S and the structures areas described previously;

wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof and wherein when Q is a sulfonamide, Q does not have a terminalreactive group or a linker arm joining the dye to a target molecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and the remaining R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹², independently comprisehydrogen, Z, a halogen, an amino group, an alkyl group wherein saidalkyl group is saturated or unsaturated, linear or branched, substitutedor unsubstituted, an alkoxy group wherein said alkoxy group is saturatedor unsaturated, branched or linear, substituted or unsubstituted, orwhen taken together R² and R³, R³ and R⁴, R⁴ and R⁵, R⁷ and R⁸, R⁸ andR⁹, R⁹ and R¹⁰, R¹ and R¹⁶, R⁶ and R¹⁸, R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁸and R¹¹, R¹³ and R¹⁴, R¹⁴ and R¹⁵, R¹⁹ and R²⁰ independently comprise afive or six membered ring;

wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and the remaining R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹², independently comprisehydrogen, Z, a halogen, an amino group, an alkyl group wherein saidalkyl group is saturated or unsaturated, linear or branched, substitutedor unsubstituted, an alkoxy group wherein said alkoxy group is saturatedor unsaturated, branched or linear, substituted or unsubstituted, orwhen taken together R² and R³, R³ and R⁴, R⁴ and R⁵, R⁷ and R⁸, R⁸ andR⁹, R⁹ and R¹⁰, R¹ and R¹⁶, R⁶ and R¹⁸, R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁸and R¹¹, R¹³ and R¹⁴, R¹⁴ and R¹⁵, R¹⁹ and R²⁰ independently comprise afive or six membered ring and the structures are as describedpreviously;

wherein Z is attached directly or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm is saturated or unsaturated, linearor branched, substituted or unsubstituted, or any combinations thereof;

and wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ or R¹²may further comprise a heteroatom containing side chain, wherein saidside chain is joined to the R group by a linkage which comprises anether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or an amine linkage(—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independentlycomprise hydrogen, Z, a halogen, an amino group, an alkyl group whereinsaid alkyl group is saturated or unsaturated, linear or branched,substituted or unsubstituted, an alkoxy group wherein said alkoxy groupis saturated or unsaturated, branched or linear, substituted orunsubstituted, or when taken together R²⁵ and R²⁶ and R²⁶ and R²⁷independently comprise a five or six membered ring, and wherein any ofR²⁵, R²⁶ or R²⁷ may further comprise said heteroatom containing sidechain.

In prior art, cyanine dyes have been disclosed that comprise an SO₃group (U.S. Pat. No. 5,268,486, U.S. Pat. No. 5,486,616 and U.S. Pat.No. 5,569,766) but the use of sulfone (SO₂) groups to modify theproperties of cyanine dyes has not been disclosed. The addition of asulfonamide group to a cyanine dye has been previously disclosed butonly in the context of being part of a linker arm (U.S. Pat. No.6,448,008) thereby being part of the connection between the dye and aterminal reactive group. Cyanine dyes lacking reactive groups, orcyanine dyes with sulfonamide groups in moieties other than the linkerarm were not disclosed in the foregoing reference.

In another aspect of the present invention, novel dyes based uponasymmetric cyanine dyes are disclosed. In one embodiment the dyes havethe structure:

wherein X comprises CR¹¹R¹², NR¹¹, O, S or Se where R¹¹ and R¹²independently comprise hydrogen, a halogen, an amino group, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkyl group is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R¹¹ and R¹² form a 5 or 6membered ring;

wherein n can be 0, 1, 2 or 3;

wherein Y is —CR⁹═CR¹⁰—;

wherein m and p can have values of 0 or 1 and m+p=1;

wherein at least one of R¹, R², R³, R⁴, R⁵, R¹¹ or R¹² comprises Q(1) orat least one of R⁶, R⁷, R⁸, R⁹ or R¹⁰ comprises Q(2);

wherein Q(1) comprises a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³),a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide(SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), aphosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonatemonoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S and the structures are as described previously;

wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof and wherein when Q is a sulfonamide, it does not comprise aterminal reactive group or a linker joining the dye to a targetmolecule;

wherein Q(2) comprises a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), asulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester(PO₃ ⁻ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) aphosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S and the structures are as described previously;

wherein Q(2) is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof and wherein when Q′ is a sulfonamide, it does not comprise aterminal reactive group or a linker joining the dye to a targetmolecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰ can be hydrogen, a halogen, an aminogroup, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup wherein said alkoxy group is saturated or unsaturated, branched orlinear, substituted or unsubstituted, or when taken together R¹³ and R¹⁴form a five or six membered ring;

wherein R¹⁶, R¹⁷, R¹⁸ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹ and R¹² can independently be hydrogen, Z, an alkyl groupwherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken together, R² and R³, R³ andR⁴, R⁴ and R⁵, R⁶ and R⁷, and R⁷ and R⁸ may form a 5 or 6 membered ring;or when taken together R¹ and R¹⁶, R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁷ andR¹⁸, and R¹⁸ and R⁹ may form a 5 or 6 membered ring;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonate monoester(PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S and the structures are as describedpreviously;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰, R¹¹ or R¹² mayfurther comprise a heteroatom containing side chain wherein said sidechain is joined to the R group by a linkage which comprises an etherlinkage (—OR²⁵), a thioether linkage (—SR²⁵), or an amine linkage(—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independentlycomprise hydrogen, Z, an alkyl group wherein said alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

In the prior art, the non-benzazolium portion of asymmetric dyes hasbeen modified with sulfonate groups (U.S. Pat. No. 5,436,134) but notthe benzazolium portion as described in the present invention.

In another aspect of the present invention, novel dyes that are basedupon xanthine dyes are disclosed. In one embodiment the dyes have thestructure:

wherein A is OR⁹ or NR¹¹R¹², where R⁹, R¹¹ and R¹² independentlycomprise hydrogen, a halogen, an amino group, an alkyl group whereinsaid alkyl group is saturated or unsaturated, linear or branched,substituted or unsubstituted, an alkoxy group wherein said alkyl groupis saturated or unsaturated, branched or linear, substituted orunsubstituted, or when taken together R¹¹ and R¹² form a five or sixmembered ring;

wherein B, when present, is O or N⁺R²¹R²² where R²¹ and R²²independently comprise hydrogen, a halogen, an amino group, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkyl group is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R²¹ and R²² form a five or sixmembered ring;

wherein C, when present, is OR²¹ or NR²¹R²² where R²¹ and R²²independently comprise hydrogen, a halogen, an amino group, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkyl group is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R²¹ and R²² form a five or sixmembered ring;

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶ R⁷, R⁸, R⁹, R¹¹, R¹², R²¹or R²² comprises Q, wherein Q comprises a sulfoxide (SOR¹³), a sulfone(SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphonate (PO₃ ⁼), aphosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S;

wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, or any combinations thereofand wherein said linker arm may be saturated or unsaturated, linear orbranched, substituted or unsubstituted or any combinations thereof andwherein when Q is a sulfonamide, it does not comprise a terminalreactive group or a linker joining the dye to a target molecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁹, R²⁰ and the remaining R¹, R², R³, R⁴, R⁵, R⁶R⁷, R⁸, R⁹, R¹¹, R¹², R²¹ or R²² can independently be hydrogen, Z, analkyl group wherein said alkyl group is saturated or unsaturated, linearor branched, substituted or unsubstituted, an alkoxy group wherein saidalkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken together R¹³ and R¹⁴, R¹ andR², R² and R⁹, R² and R¹¹, R⁹ and R³, R¹¹ and R³, R⁴ and R²¹, R²¹ andR⁵, R⁶ and R⁷, R⁶ and R⁷, or R⁸ and R¹ form a five or six membered ring;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonate monoester(PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

wherein any of R¹, R², R³, R⁴, R⁵, R⁶ R⁷, R⁸, R⁹, R¹¹, R¹², R²¹ or R²²may further comprise a heteroatom containing side chain wherein saidside chain is joined to the R group by a linkage which comprises anether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or an amine linkage(—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independentlycomprise hydrogen, Z, an alkyl group wherein said alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

Coumarin

In another aspect of the present invention, novel dyes that are basedupon coumarin dyes are disclosed. In one embodiment the dyes have thestructure:

wherein R⁷ comprises an amine group, a hydroxyl group, or a moiety thatcan enzymatically be converted into an amine group or hydroxyl group;

wherein at least one of R³, R⁴, R⁵, R⁶, or R⁸ comprises Q, wherein Qcomprises a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide(SO₂NR¹³R¹⁴), a phosphate monoester (PO₃ ⁻ER¹³), a phosphate diester(PO₂ER¹³ER¹⁴), a phosphonate monoester (PO₂ ⁻ER¹³), a phosphonatediester (POER¹³ER¹⁴), a thiophosphate (PSO₃ ⁼), a thiophosphatemonoester (PSO₂ ⁻ER¹³), a thiophosphate diester (PSOER¹³ER¹⁴), athiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO⁻ER¹³), athiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴), or its thioanalogue (POSR¹⁹NR¹³R¹⁴),wherein any of E independently comprises O or S;

wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, or any combinations thereofand wherein said linker arm may be saturated or unsaturated, linear orbranched, substituted or unsubstituted or any combinations thereof andwherein when Q is a sulfonamide, Q does not have a terminal reactivegroup or a linker arm joining the dye to a target molecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and the remaining R³, R⁴,R⁵, R⁶, or R⁸ independently comprise hydrogen, Z, a halogen, an aminogroup, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup wherein said alkoxy group is saturated or unsaturated, branched orlinear, substituted or unsubstituted, or when taken together R³ and R⁴,R⁴ and R⁵, and R⁵ and R⁶ independently comprise a five or six memberedring;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), a phosphonate monoester(PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³), a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴), or its thioanalogue (POSR¹⁹NR¹³R¹⁴),wherein any of E independently comprises O or S;

wherein Z is attached directly or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, or any combinations thereofand wherein said linker arm is saturated or unsaturated, linear orbranched, substituted or unsubstituted, or any combinations thereof;

and wherein any of R³, R⁴, R⁵, R⁶, or R⁸ may further comprise aheteroatom containing side chain, wherein said side chain is joined tothe R group by a linkage which comprises an ether linkage (—OR²⁵), athioether linkage (—SR²⁵), or an amine linkage (—NR²⁵R²⁶ or—N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independently comprisehydrogen, Z, a halogen, an amino group, an alkyl group wherein saidalkyl group is saturated or unsaturated, linear or branched, substitutedor unsubstituted, an alkoxy group wherein said alkoxy group is saturatedor unsaturated, branched or linear, substituted or unsubstituted, orwhen taken together R²⁵ and R²⁶ and R²⁶ and R²⁷ independently comprise afive or six membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ mayfurther comprise said heteroatom containing side chain.

Particularly useful varieties of the novel dyes of the present inventionmay be based upon 6,8 difluoro-7-hydroxycoumarin as described in U.S.Pat. No. 5,830,912 incorporated herein by reference.

In another aspect of the present invention, novel dyes that are basedupon styrene dyes are disclosed. In one embodiment the dyes have thestructure:

wherein X comprises CR¹¹R¹², NR¹¹, O, S or Se where R¹¹ and R¹²independently comprise hydrogen, a halogen, an amino group, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkyl group is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R¹¹ and R¹² form a 5 or 6membered ring;

wherein n can be 1, 2 or 3;

wherein at least one of of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹²,R²¹ or R²² comprises Q, wherein Q comprises a sulfoxide (SOR¹³), asulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphonate (PO₃⁼), a phosphonate monoester (PO₂ ⁻ER¹³) a phosphonate diester(POER¹³ER¹⁴), a thiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂⁻ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂⁼), a thiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S;

wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, or any combinations thereofand wherein said linker arm may be saturated or unsaturated, linear orbranched, substituted or unsubstituted or any combinations thereof andwherein when Q(2) is a sulfonamide, it does not comprise a terminalreactive group or a linker joining the dye to a target molecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰ can be hydrogen, a halogen, an aminogroup, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup wherein said alkoxy group is saturated or unsaturated, branched orlinear, substituted or unsubstituted, or when taken together R¹³ and R¹⁴form a five or six membered ring;

wherein R¹⁶, R¹⁷ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹¹, R¹²R¹⁶, R¹⁷, R²¹ and R²² can independently be hydrogen, Z, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken together, R² and R³, R³ andR⁴, R⁴ and R⁵, R⁶ and R⁷, R⁷ and R⁸, R¹ and R¹⁶, R¹¹ and R¹⁶, R¹⁶ andR¹⁷, R¹⁷ and R¹⁸, and R¹⁸ and R⁹ may form a 5 or 6 membered ring;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonate monoester(PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

and wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹², R²¹ orR²² may further comprise a heteroatom containing side chain wherein saidside chain is joined to the R group by a linkage which comprises anether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or an amine linkage(—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independentlycomprise hydrogen, Z, an alkyl group wherein said alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

In another embodiment of the present invention, the styryl dye comprisesa picoline or quinoline moiety instead of a benzazolium group. As such,these dyes have the structure:

wherein n can be 1, 2 or 3;

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R²¹ or R²²comprises Q wherein Q comprises a sulfonate (SO₃ ⁻), a sulfonate ester(SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide(SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), aphosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonatemonoester (PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³) a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) where E can be independently O or S;

wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, or any combinations thereofand wherein said linker arm may be saturated or unsaturated, linear orbranched, substituted or unsubstituted or any combinations thereof andwherein when Q is a sulfonamide, it does not comprise a terminalreactive group or a linker joining the dye to a target molecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰ can be hydrogen, a halogen, an aminogroup, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup wherein said alkoxy group is saturated or unsaturated, branched orlinear, substituted or unsubstituted, or when taken together R¹³ and R¹⁴form a five or six membered ring;

wherein R¹⁶, R¹⁷ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R²¹ or R²² can independently be hydrogen, Z, an alkyl group wherein saidalkyl group is saturated or unsaturated, linear or branched, substitutedor unsubstituted, an alkoxy group wherein said alkoxy group is saturatedor unsaturated, branched or linear, substituted or unsubstituted, orwhen taken together, R¹ and R², R² and R³, R³ and R⁴, R⁴ and R⁵, R⁵ andR¹⁶, R¹⁶ and R¹⁷, R¹⁷ and R⁹, R⁹ and R⁸, R⁸ and R²¹, R²¹ and R²², R²²and R⁷, and R⁷ and R⁶ may form a 5 or 6 membered ring;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼) a phosphonate monoester(PO₂ ⁻ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³) a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

and wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R²¹ or R²² mayfurther comprise a heteroatom containing side chain wherein said sidechain is joined to the R group by a linkage which comprises an etherlinkage (—OR²⁵), a thioether linkage (—SR²⁵), or an amine linkage(—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independentlycomprise hydrogen, Z, an alkyl group wherein said alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

When R⁴ and R⁵ comprise alkyl chains that are joined together, aquinoline moiety can be formed, the dye thereby having the structure:

Where R⁴¹, R⁴², R⁴³ and R⁴⁴ are as described previously for R¹, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R²¹ and R²².Complex Ring Structures

As described above some of the R groups may be joined together to formone or more fused 5 or 6 membered ring structures. It is understood thatthe complex rings that are formed by closure of R groups may be furthersubstituted with any of the R groups described previously. Examples ofcomplex rings that may be formed for the benzazolium portion of cyanineand asymmetric cyanine dyes can comprise but not be limited to:

In addition, “rigid” cyanine dyes have been described where a fused ringis formed where the nitrogen of the benzazolium is linked to the nearestcarbon of the methine bridge (U.S. Pat. No. 6,133,445 and U.S. Pat. No.6,686,145 both of which are hereby incorporated by reference). Similarlyin a cyanine dye with a monomethine bridge (i.e. when n=0), a rigidlinkage can be formed by joining the nitrogens of the benzazolium groupto each other (U.S. Pat. No. 5,852,191 and U.S. Pat. No. 5,981,747 bothof which are incorporated by reference).

If desired, a variation of the preceding dyes can be the substitution ofan azabenzazolium instead of a benzazolium moiety in the cyanine,asymmetric cyanine and styrene dyes; i.e. a Nitrogen replaces the carbonin the positions where R², R³, R⁴, R⁵, R⁷, R⁸, R⁹ or R¹⁰ are connectedto the benzazolium moiety of cyanine dyes or to the R², R³, R⁴ or R⁵positions of the asymmetric cyanine and styrene dyes disclosedpreviously. Methods for the synthesis and use of an azabenzazolium baseddyes are disclosed in U.S. Pat. No. 6,664,047 B1, hereby incorporated byreference. As such these moieties would have the structures:

Examples of rings and complex rings that may comprise thenon-benzazolium portion of an asymmetric cyanine dye can comprise butnot be limited to:

Examples of rings and complex rings that may be part of thenon-benzazolium portion of a styryl dye can comprise but not be limitedto:

Reactive Groups and Targets

In another aspect of the present invention, one of the R groups is areactive group thereby allowing the dyes of the present invention to beattached to a useful target molecule. Examples of reactive groups thatmay find use in the present invention can include but not be limited toa nucleophilic reactive group, an electrophilic reactive group, aterminal alkene, a terminal alkyne, a platinum coordinate group or analkylating agent.

There are a number of different electrophilic reactive groups that mayfind use with the present invention; examples can include but not belimited to isocyanate, isothiocyanate, monochlorotriazine,dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogensubstituted pyridine, mono- or di-halogen substituted diazine,maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-proprionamide,glyoxal and aldehyde groups. Nucleophilic reactive groups can includebut not be limited to reactive thiol, amine and hydroxyl groups. Forpurposes of synthesis of dyes, reactive thiol, amine or hydroxyl groupscan be protected during various synthetic steps and the reactive groupsgenerated after removal of the protective group. Use of a terminalalkene or alkyne groups for attachment of markers has been previouslydescribed in U.S. Patent Application Ser. No. 2003/0225247, herebyincorporated by reference. The use of platinum coordinate groups forattachment of other dyes has been previously disclosed in U.S. Pat. No.5,580,990 and the use of alkyl groups has been previously described inU.S. Pat. No. 6,593,465 both of which are hereby incorporated byreference.

Examples of useful target molecules can include but not be limited to anucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleicacid, protein, peptide, enzyme, antigen, antibody, hormone, hormonereceptor, cellular receptor, lymphokine, cytokine, hapten, lectin,avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide,polysaccharide, lipid, glycolipid, viral particle, viral component,bacterial cell, bacterial component, eucaryotic cell, eukaryotic cellcomponent, natural drug, synthetic drug, glass particle, glass surface,natural polymers, synthetic polymers, plastic particle, plastic surface,silicaceous particle, silicaceous surface, organic molecule, dyes andderivatives thereof.

The nucleoside, nucleotide, oligonucleotide, or polynucleotide cancomprise one or more ribonucleoside moieties, ribonucleotide moieties,deoxyribonucleoside moieties, deoxyribonucleotide moieties, modifiedribonucleosides, modified ribonucleotides, modifieddeoxyribonucleosides, modified deoxyribonucleotides, ribonucleotideanalogues, deoxyribonucleotide analogues and any combination thereof.

As described above, the dyes of the present invention may have dyes astargets thereby creating composite dyes. By joining the dyes of thepresent invention to another dye, unique properties may be enjoyed thatare not present in either dye alone. For instance, if one of the dyes ofthe present invention is joined to another dye such that it creates anextended conjugation system, the spectral characteristics of the dye maybe different than either dye component. Another example of this methodis where the conjugation systems do not overlap but the proximity allowsan internal energy transfer to take place thereby extending the Stokesshift. For an example of this, see U.S. Pat. No. 5,401,847, U.S. Pat.No. 6,008,373 and U.S. Pat. No. 5,800,996 all of which are herebyincorporated by reference. Other properties may also be enhance by thisjoining, for example, it has been previously described that the joiningtogether of two ethidium bromide molecules generates a dye that hasenhanced binding to nucleic acids (U.S. Patent Application Serial No.2003/0225247, hereby incorporated by reference). Other composite dyeshave been described that simultaneously enjoy both properties, i.e.enhanced binding and energy transfer (U.S. Pat. No. 5,646,264 herebyincorporated by reference). Furthermore, these composites dyes are notlimited to binary constructs of only two dyes, but may compriseoligomeric or polymeric dyes. These composite dyes may be comprised ofthe same dye or different dyes may be joined together depending upon theproperties desired.

Utility may also be achieved by attaching a dye of the present inventionto a target specific moiety. Thus, binding between the target specificmoiety and its corresponding target may be monitored by essentiallydetermining the presence or amount of dye that is bound to the target.Well-known examples of such assays are hybridizations betweencomplementary nucleic acids as well as binding that takes place betweenantibodies and their corresponding antigens. Other binding pairs thatmay be of interest can include but not be limited to ligand/receptor,hormone/hormone receptor, carbohydrate/lectin and enzyme/substrate.Assays may be carried out where one component is fixed to a solidsupport and a corresponding partner is in solution. By binding to thecomponent fixed to the support, the partner now becomes attached to thesupport as well. A well-known example of this method is the microarrayassays where labeled analytes become bound to discrete sites on themicroarray. Homogeneous probe dependent assays are also well known inthe art and may take advantage of the present invention. Examples ofsuch methods are energy transfer between adjacent probes (U.S. Pat. No.4,868,103), the Taqman exonuclease assay (U.S. Pat. No. 5,538,848 andU.S. Pat. No. 5,210,015), Molecular Beacons (U.S. Pat. No. 5,118,801 andU.S. Pat. No. 5,925,517) and various real time assays (U.S. patentapplication Ser. No. 10/096,076), all of which are incorporated byreference.

Antibodies labeled with dyes of the present invention may be used invarious formats. For example, an antibody with one of the dyes of thepresent invention may be used in an immunofluorescent plate assay or insitu analysis of the cellular location and quantity of various antigenictargets. Antibodies labeled with dyes may also be used free in solutionin cell counting or cell sorting methods that use a flow cytometer.

The presence or absence of a signal may then be used to indicate thepresence or absence of the target itself. An example of this is a testwhere it is sufficient to know whether a particular pathogen is presentin a clinical specimen. On the other hand, quantitative assays may alsobe carried out where it is not so much the intention of evaluating if atarget is present but rather the particular amount of target that ispresent. An example of this is the previously cited microarray assaywhere the particular rise or fall in the amount of particular mRNAspecies may be of interest.

In another embodiment of the present invention, dyes that have beendisclosed above as well as dyes described previous literature may beattached to a carrier with a more general affinity. Dyes may be attachedto intercalators that in themselves do not provide signal generation butby virtue of their binding may bring a dye in proximity to a nucleicacid. A further example is attachment of dyes to SDS molecules therebyallowing dyes to be brought into proximity to proteins. Thus thisembodiment describes the adaptation of a dye or dyes that lack affinityto a general class of molecules may be adapted by linking them tonon-dye molecules or macromolecules that can convey such properties.

The dyes of the present invention may also be used without tetheringthem to a target specific moiety. For example, it has long been knownthat ethidium bromide has a high affinity for binding to double-strandedDNA. For many years this has been a standard method of identifying andcharacterizing nucleic acids that have been separated by molecularweights after electrophoresis in a gel. An additional useful property ofethidium bromide that has been exploited in such gel assays is theheightened fluorescence that takes place after ethidium has bound to adouble-stranded nucleic acid molecule. A similar effect has been seenfor stains that can increase fluorescence or even change emissionwavelengths after binding to proteins (See Chapter 9.1 in “Handbook ofMolecular Probes and Research Chemicals” 6^(th) edition, 1996, MolecularProbes, Inc. Eugene, Oreg., incorporated hereby by reference). Thesestains may be relatively specific for particular macromolecules or theymay have a more general affinity. For instance a cyanine based dye“Stains all” has been used for staining nucleic acids, proteins,polysaccharides and lipids (Green 1975 J Histochem Cytochem 23; 411-423,Kelly and Parker, 1981 J Bact 145; 1018-1024). The effects ofinteractions of this dye can be seen by the production of various colorsdepending upon the nature of the substance bound to the dye i.e. DNA isblue, RNA is bluish purple and proteins are generally pink or reddishalthough there are variations for individual proteins.

Various applications may enjoy the benefits of binding the dyes of thepresent invention to appropriate targets. As described above, stainingof macromolecules in a gel is a methodology that has a long history ofuse. More recent applications that also may find use are real timedetection of amplification (U.S. Pat. No. 5,994,056, U.S. Pat. No.6,174,670 and U.S. patent application Ser. No. 10/096,076, all of whichare hereby incorporated by reference), and binding of nucleic acids tomicroarrays. In situ assays may also find use where the binding of dyesof the present invention is used to identify the location or quantity ofappropriate targets.

It is also a subject of the present invention that the dyes of thepresent invention as well as dyes that have been described previously inthe literature may be joined together to create composite dyes withmultiple binding properties. For instance, a dye that has an affinityfor nucleic acids can be joined to a dye that has an affinity forproteins, thereby creating a composite dye that has an affinity for bothnucleic acids and proteins. If the particular affinities of these dyesare strong, the multiple binding dye will have an expanded range of usecontributed by the properties of each of the individual affinities. Thusfor instance, a dye with an affinity for nucleic acids and a lack ofaffinity for proteins may be joined to a dye with an affinity forproteins and a lack of affinity for nucleic acids to create a multiplebinding dye with affinities for both nucleic acids and proteins. On theother hand, two dyes may be chosen that each have a discrete but lowaffinity for their targets, where the composite dye has an overallhigher affinity of binding (i.e cooperative binding) when each of thetargets is in proximity to each other. In the present invention, thetargets are comprised of different types of macromolecules, and as suchthe components of the composite dyes have heterogeneous affinities. Thusfor example, a dye that has an affinity for lipids joined to a dye thathas an affinity for proteins may create a co-operative binding dye thathas a higher affinity for proteins in cell membranes. In anotherexample, a dye with an affinity for proteins joined to a dye with anaffinity for nucleic acids may create a co-operative binding dye with aspecial affinity for proteins associated with or bound to nucleic acids.In a preferred embodiment, one or both dyes have enhanced fluorescenceupon binding to their appropriate targets. The detection of binding ofsuch dyes can enjoy energy transfer, quenching, changes in wavelength oreven a mixture of color emissions.

It should be pointed out that a composite product with affinities fortwo different groups does not need to have dyes in each of thecomponents. The major principle is that the binding event is dependentupon the simultaneous presence of both targets. Thus for example, anon-fluorescent compound that binds to a particular target can becombined with a fluorescent dye that has an affinity for a differenttarget and resultant co-operative binding dye can exhibit enhancedsignaling when each of the targets are in proximity to each other.Furthermore, in addition to being neutral in terms of having an absenceof fluorescent signal capability, one of the partners may be of anegative nature, i.e a fluorescence quencher. Thus for instance, whenthe multiple binding dye is in free in solution due to a lack oftargets, the quencher in one component effectively inhibits fluorescencein the other component. However, simultaneous binding of the dye to eachof the different target types can result in a loss of the quenching. Anexample of a system with a single binding affinity that has beenpreviously mentioned is the Molecular Beacon method, where binding ofthe probe to its appropriate target causes a physical separation of aquencher and a fluorescent moiety thereby generating a binding dependentsignal.

It should also be understood that it is not a necessity for each of thecomponents to have a single affinity thereby creating a compositemultiple binding dye with only two affinities. Again, to use theexamples described previously, a dye with a high affinity for nucleicacids and a low affinity for lipids can be joined to a dye with a highaffinity for proteins to produce a multiple binding dye with highaffinities for both proteins and nucleic acids. In a similar fashion, adye with a low affinity for nucleic acids may be joined to a dye with alow affinity for both proteins and lipids thereby creating a multiplebinding dye that may have a cooperative affinity for bothprotein/nucleic acid complexes and for nucleic acid/membrane complexes.

The examples which follow are set forth to illustrate various aspects ofthe present invention but are not intended in any way to limit its scopeas more particularly set forth and defined in the claims that followthereafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 Synthesis of Dye 101(a) Preparation of 7-Formyl julolidine (Compound 1)

A solution of julolidine (15.0 g, 86.6 mmol) in DMF (100 ml) was cooledin an ice bath. While vigorously stirring this solution, POCl₃ (20.0 g,129.8 mmol) was added drop wise. After the addition of POCl₃ wascomplete, the ice bath was removed and the reaction mixture was stirredat room temperature for 30 minutes. An aqueous solution of sodiumacetate (25% w/w, 20 ml) was then added to the reaction mixture and itwas heated in an oil bath (T=110° C.) for 10 min. The reaction mixturewas cooled and poured into ca. 500 ml water and extracted with ethylacetate. The organic layer was washed twice with water followed bybrine, dried over sodium sulfate and then evaporated to dryness to yield12.0 g of a light yellow solid (Compound 1) with the structure givenbelow:

(b) Preparation of 2-methylbenzothiazole-6-sulfonyl chloride (Compound2)

Chlorosulfonic acid (20 ml, 335.0 mmol) was cooled in an ice bath and2-methylbenzothiazole (10.0 g, 67.0 mmol) was carefully added drop wiseover a period of 30 minutes. The combined mixture was heated at 115-120°C. for 15 hours and after cooling, the mixture was added very slowly toca. 200 ml ice/water mix. A sticky white solid separated which wasextracted into chloroform (300 ml). The organic layer was washed withwater (2×, 350 ml), washed with brine (2×, 350 ml), dried and evaporatedto yield 11.2 g (67%) of a colorless oil (Compound 2) which solidifiedupon cooling, R_(f)=0.5 (30% ethyl acetate in hexane). The structure ofCompound 2 is given below:

(c) Preparation of N-(2-methylbenzothiazole-6-sulfonyl)piperidine(Compound 3)

A solution of Compound 2 (2.5 g, 10 mmol) in chloroform (15 ml) wasadded drop wise to a solution of piperidine (1.7 g, 20.0 mmol) inchloroform (10 ml). The combined mixture was stirred at room temperaturefor 1 hour. The reaction mixture was then washed with water (2×, 50 ml)and brine (1×, 50 ml). The organic layer was dried with sodium sulfateand evaporated. The sticky white solid thus obtained was dissolved inca. 5 ml of hot ethyl acetate and this solution was then slowly added to40 ml hexane. The white solid that precipitated was collected, washedwith hexane and dried to yield 2.2 g (74%) of Compound 3, R_(f)=0.28(30% ethyl acetate in hexane). The structure of Compound 3 is givenbelow:

(d) Preparation of N-(2,3-dimethylbenzothiazole-6-sulfonyl)piperidinetosylate (Compound 4)

A mixture of Compound 3 (1.0 g, 3.4 mmol) and p-toluenesulfonic acidmethyl ester (0.94 g, 5.1 mmol) was heated in a pressure tube at 130° C.for 1 hour. The mixture was allowed to cool to room temperature, and theresulting mass was triturated with acetone (25 ml) until a gray coloredsolid separated. The solid was collected by centrifugation, washed withacetone and dried under vacuum to yield 1.4 g (86%) of Compound 4 whosestructure is given below:

(e) Preparation of Dye 101

A mixture of Compound 1 (0.19 g, 0.93 mmol), Compound 4 (0.30 g, 0.62mmol) and piperidine (92 μL, 0.93 mmol) was heated in glacial aceticacid (3 ml) at 115-120° C. for 2 hours. The reaction mixture was cooledto room temperature and mixed with 30 ml isopropanol. This combinedmixture was added drop wise to 300 ml hexane and the dark purple solidprecipitate was collected and dried to yield 0.4 g (yield: 96%) of Dye101. Abs (max, in methanol)=588 nm; Em=624 nm, ε=90,580 M⁻¹cm⁻¹. Thestructure of this dye is as follows:

Example 2 Synthesis of Dye 102 (a) Preparation of N-(6-carboxylhexane)-2,3,4 tetrahydro quinoline (Compound 5)

A mixture of 1,2,3,4 tetrahydro quinoline (10.0 g, 75.0 mmol),6-bromohexanoic acid (21.9 g, 112.5 mmol) and triethyl amine (11.4 g,112.5 mmol) in 50 ml ethanol was refluxed for 16 hours. The reactionmixture was cooled and precipitated. The precipitate was then collectedby filtration. The remaining solvents were removed in a rotaryevaporator and to the residue thus obtained 200 ml ethyl acetate and 200ml water was added. The organic layer was separated, washed with waterand brine, dried over sodium sulfate and evaporated to dryness to yield20.0 g of a dark brown oil (Compound 5) with the structure given below.

(b) Preparation of N-(6-carboxyl hexane)-2,3,4 tetrahydro-7-formylquinoline (Compound 6)

This procedure was carried out as described previously in step (a) ofExample 1, with POCl₃ (4.7 g, 30.3 mmol) and DMF (40 ml) and usingcompound Compound 5 (5.0 g, 20.7 mmol) instead of the julolidine used instep (a) of Example 1. Compound 6 was obtained as a dark liquid (3.46 g)and used without any further purification. The structure of Compound 6is given below:

(c) Preparation of Dye 102

The procedure was carried out as described previously in step (e) ofExample 1 with Compound 4 (0.24 g, 0.5 mmol), piperidine (72 μL, 0.73mmol) and glacial acetic acid (2 ml) and substituting Compound 6 (0.2 g,0.73 mmol) in place of the Compound 1 used in Example 1. The resultantDye 102 was obtained as a dark purple solid (0.25 g, yield: 68%). Thestructure of Dye 102 is given below:

Example 3 Synthesis of Dye 103 (a) Preparation ofN—[N′-(4′-Sulfobutyl)-2-methylbenzothiazole-6-sulfonyl)piperidine, InnerSalt (Compound 7)

A mixture of Compound 3 (1.0 g, 3.4 mmol) (from step (c) of Example 1)and 1-4-butane sultone (0.55 g, 4.0 mmol) was heated in a pressure tubeat 140-145° C. for 3 hours. The mixture was allowed to cool to roomtemperature, and the resulting mass was triturated with acetone (40 ml)until a pink solid separated. The solid was collected by centrifugation,washed with acetone and dried under vacuum to yield 0.21 g (14%) ofCompound 7 with the structure:

(b) Preparation of Dye 103

A mixture of compound 7 (0.20 g, 0.46 mmol),9-ethyl-3-carbazolecarboxaldehyde (0.11 g, 0.50 mmol) and piperidine (20μL, 0.2 mmol) was refluxed in ethanol (2 ml) for 18 hours. The reactionmixture was cooled to room temperature and mixed with 20 ml ethylacetate. The precipitated solid was collected by centrifugation, washedwith ethyl acetate (2×25 ml) and dried under vacuum to yield 0.27 g ofan orange brown solid (Dye 103). For spectral analysis, a small amount(˜40 mg) of Dye 103 was purified on a silica column using a stepwisegradient of methanol (5% to 15%) in chloroform. Abs (max, methanol)=503nm; Em=596 nm, ε=30,000 M⁻¹cm⁻¹. The structure of Dye 103 is givenbelow:

Example 4 Synthesis of Dye 104

This process was carried out as described previously in step (b) ofExample 3, using 9-ethyl-3-carbazolecarboxaldehyde (0.10 g, 0.45 mmol),piperidine (18 μL, 0.18 mmol) and ethanol (3 ml) but in this procedure,Compound 4 (0.2 g, 0.41 mmol) from step (d) of Example 1 was usedinstead of the Compound 7 used in Example 3. The resultant Dye 104 wasprecipitated using ethyl ether, as a brown solid (0.23 g, yield: 81%).The structure of this dye is given below:

Example 5 Synthesis of Dye 105 and its NHS Ester (a) Preparation ofIntermediate

A mixture of Compound 4 (1.0 g, 2.1 mmol) from step (d) of Example 1 andN, N′-diphenylformamidine (0.5 g, 2.5 mmol) in acetic acid (5 ml) washeated and refluxed for 2 hours. The reaction mixture was cooled andadded to ethyl ether (45 ml). The precipitated dark colored solid wascollected by centrifugation, washed with ether, dried under Argon andimmediately used in the next step.

(b) Preparation of Dye 105

The intermediate obtained in step (a) was added to a solution of1-(ε-carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate (1.3 g,3.6 mmol) dissolved in a mixture of acetic anhydride (10 ml) andpyridine (10 ml). The combined mixture was stirred in the dark at roomtemperature for 18 hours. The precipitated solid was collected bycentrifugation, washed with a pyridine/acetic anhydride mixture (1:1, 30ml), washed with ethyl acetate (2×, 30 ml) and dried to yield 0.63 g ofa purple solid (Dye 105). Abs (max, in water)=567 nm; Em=580 nm. Thestructure of Dye 105 is given below:

(c) Preparation of NHS Ester of Dye 105

A mixture of Dye 105 (0.5 g, 0.74 mmol), N-hydroxysuccinimide (0.13 g,1.11 mmol) and DCC (0.23 g, 1.11 mmol) in DMF (2 ml) was stirred at roomtemperature in the dark for 17 hours. DCC-urea precipitated out and wasremoved by centrifugation. The supernatant was added drop wise to a10-fold excess of ethyl acetate. The precipitated dark solid wascollected by centrifugation, washed with ethyl acetate and dried toyield 80.0 mg of the NHS ester of Dye 105. The structure of thiscompound is given below:

Example 6 Synthesis of Benzoxazole Dyes

The previous examples have been based upon a benzothiazole moiety. Avariety of different dyes may also be synthesized that are based upon abenzoxazole moiety instead.

(a) Preparation of 2-methylbenzoxazole-6-sulfonyl chloride (Compound 8)

Chlorosulfonic acid (12.5 ml, 187.8 mmol) was cooled in an ice bath and2-methylbenzoxazole (5.0 g, 37.5 mmol) was carefully added drop wiseover a period of 15 min. The combined mixture was heated at 115-120° C.for 16 hours. After cooling, the mixture was added very slowly to ca.100 ml ice/water mix. The white solid that separated was collected byfiltration, washed with water until washings were neutral and dried toyield 5.2 g (59%) of Compound 8, whose structure is given below:

(b) Preparation of N-(2-methylbenzoxazole-6-sulfonyl)piperidine(Compound 9)

Piperidine (2.2 g, 26 mmol) was added drop wise to a solution ofCompound 8 (3.0 g, 10 mmol) in chloroform (30 ml) and DMF (10 ml). Thecombined mixture was stirred at room temperature for 1 hour, and thenwashed with water (2×, 50 ml) and brine (1×, 50 ml). The organic layerwas then dried over sodium sulfate and evaporated to provide 2.76 g(76%) of Compound 9 as a sticky white solid. TLC analysis showedR_(f)=0.25 (30% ethyl acetate in hexane). The structure of this compoundis given below:

Compound 9 may now be used in the same processes that used (Compound 3)as a reagent to make benzoxazole analogues of Dye 101, Dye 102, Dye 103,Dye 104, Dye 105 as well as other dyes.

Example 7 Synthesis of Dye 106 (NHS Ester of CX2) (a) Preparation ofCompound 10

A mixture of 4-methyl pyridine (20 ml) and 6-bromohexanoic acid (10 g)was refluxed overnight. The mixture was cooled to room temperature, and20 ml of acetone was added. The mixture was stirred at room temperaturefor 15 minutes followed by addition of 80 ml ether. After filtration, 18g of a yellow solid (Compound 10) was collected. The structure ofCompound 10 is given below:

(b) Preparation of Dye 106 from Compound 10

To a solution of 2 g of 1-ethyl-2,3,3,-trimethylindolinium 5-sulfatedissolved in acetic acid (40 ml), 1.6 g of N,N-diphenylformamide wasadded. The reaction mixture was refluxed for 4 hours and then added dropwise into a stirring mixture of ethyl acetate (25 ml) and ether (25 ml)under argon. The resultant precipitate was collected by centrifugationand added to a solution of 2 g of Compound 10 (prepared in step (a))dissolved in a mixture of 8 ml of pyridine and 8 ml of acetic anhydride.The reaction mixture was heated to 60-70° C. overnight under argon.After cooling to room temperature, the reaction mixture was added to 200ml of ethyl acetate. The solution was filtered and the solid residue wasdissolved in 50 ml of dry DMF. The DMF was evaporated in vacuum and theresidue was dissolved in 50 ml of dry DMF again. To the above solution,1,3-dicyclohexyl-carbodiimide (5 g) and N-hydroxysuccinimide (5 g) wasadded. The reaction mixture was stirred at room temperature overnight.Filtration was then used to remove precipitated byproducts. The solutionwas concentrated by rotary evaporation and the product was purified bysilica gel chromatography eluted with 20% methanol in methyl chloride togive 600 mg of a black solid (Dye 106) whose structure is given below:

Example 8 Preparation of UTP Labeled with Dye 106

A solution of 58.1 mg of Dye 106 (from Example 7) dissolved in 1 ml ofDMF was added to a mixture of allylamine modified UTP (200 μl of 50μmol/ml solution), KHCO₃ (50 mg), 50 μl of 7M LiCl and 5 ml of water.The reaction mixture was stirred at room temperature overnight. Afteraddition of 10 ml of water to the reaction mixture, the solution wasextracted with n-BuOH (3×20 ml). The aqueous layer was collected andloaded onto a DEAE-Sephadex column and eluted with a 0.1 M to 0.9 M TEABgradient. Fractions were checked by HPLC and the appropriate fractionswere pooled to give 17 μmol of UTP labeled with Dye 106. The structureof the final product is given below:

Example 9 Synthesis of Dye 107 (NHS Ester of CX1) (a) Preparation ofCompound 11

Preparation of this compound was carried out essentially as described instep (a) of Example 7 except that 4-methyl quinoline was substituted forthe 4-methyl pyridine used in Example 7. The resultant product (Compound11) has the following structure:

(b) Preparation of Dye 107 Using Compound 11

To a solution of 8 g of 1-ethyl-2,3,3,-trimethylindolinium 5-sulfonatedissolved in acetic acid (40 ml), 6.6 g of N,N-diphenylformamide wasadded. The reaction mixture was refluxed for 4 hours and then addeddropwise into a stirring mixture of ethyl acetate (150 ml) and ether(150 ml) under argon. The precipitate was collected by centrifugationand added to a solution of 7 g of Compound 11 from step (a) dissolved ina mixture of 50 ml of pyridine and 20 ml of acetic anhydride. Thereaction mixture was heated to 60-70° C. for 2 hours under argon. Aftercooling to room temperature, the reaction mixture was added to 600 ml ofethyl acetate. The solution was filtered and the solid residue wasdissolved in 200 ml of dry DMF. The DMF was evaporated in vacuum and theresidue was dissolved in 100 ml of dry DMF again. To the above solution,1,3-dicyclohexyl-carbodiimide (8.86 g) and N-hydroxysuccinimide (10 g)was added. The reaction mixture was stirred at room temperatureovernight. After filtration, the solvent was evaporated and the residuewas separated by silica gel chromatography eluted with 20% methanol inmethyl chloride to give 3.2 g of a black solid (Dye 107) whose structureis given below:

Example 10 Preparation of UTP Labeled with Dye 107

A solution of 62.9 mg of Dye 107 (from Example 9) dissolved in 1 ml ofDMF was added to a mixture of allylamine modified UTP (200 μl of 50μmol/ml solution), KHCO₃ (50 mg), 50 μl of 7M LiCl and 5 ml of water.The reaction mixture was stirred at room temperature overnight. Afteraddition of 10 ml of water to the reaction mixture, the solution wasextracted with n-BuOH (3×20 ml). The aqueous layer was collected andloaded onto a DEAE-Sephadex column and eluted with a 0.1 M to 0.9 M TEABgradient. Fractions were checked by HPLC and the appropriate fractionswere pooled to give 45.6 μmol of UTP labeled with Dye 107. The structureof the final product is given below:

Example 11 Synthesis of Dye 108 (NHS Ester of CX3) a) Preparation ofCompound 12

A mixture of potassium salt of indoleninium sulfonate (8.0 g, 28.8 mmol)and 6-bromohexanoic acid (7.32 g, 37.5 mmol) were heated in a pressuretube at 120-125° C. for 16 hours. The resulting mass was dissolved inboiling DMF (20 mL) and this solution was drop wise added to 200 mLethyl acetate. The pink solid that precipitated was collected bycentrifugation, washed with ethyl acetate and dried under vacuum toyield 21.6 g (89%) of CD-11 with the structure:

(b) Preparation of Compound 13

Compound 13 was prepared by refluxing ethyl iodide with methyl6-methylnicotinate. The structure of Compound 13 is shown below:

(c) Preparation of Dye 108 Using Compound 12 and Compound 13

To a solution of 1 g of Compound 12 from step (a) dissolved in aceticacid (6 ml), 1 g of N,N-diphenylformamide was added. The reactionmixture was refluxed for 4 hours and then added dropwise into a stirringmixture of ethyl acetate (20 ml) and ether (20 ml) under argon. Theprecipitate was collected by centrifugation and added to a solution 1.5g of Compound 13 from step (b) dissolved in a mixture of 10 ml ofpyridine and 10 ml of acetic anhydride. The reaction mixture was heatedto 60-70° C. for 2 hours under argon. After cooling to room temperature,the reaction mixture was added to 200 ml of ethyl acetate, The solutionwas filtered and the solid residue was dissolved in 200 ml of dry DMF.The DMF was evaporated in vacuum and the residue was dissolved in 100 mlof dry DMF again. To the above solution, 1,3-dicyclohexyl-carbodiimide(3 g) and N-hydroxysuccinimide (4 g) were added. The reaction mixturewas stirred at room temperature overnight. After filtration, the solventwas evaporated and the residue was separated by silica gelchromatography eluted with 20% methanol in methyl chloride to give 0.4 gof a black solid (Dye 107) whose structure is given below.

Example 12 Preparation of UTP Labeled with Dye 108

A solution of 100 mg of Dye 107 (from Example 10) dissolved in 5 ml ofDMF was added to a mixture of allylamine modified UTP (200 μl of 50μmol/ml solution), KHCO₃ (20 mg), 200 μl of 7M LiCl and 5 ml of water.The reaction mixture was stirred at room temperature overnight. Afteraddition of 10 ml of water to the reaction mixture, the solution wasextracted with n-BuOH (3×20 ml). The aqueous layer was collected andloaded onto a DEAE-Sephadex column and eluted with a 0.1 M to 0.9 M TEABgradient. Fractions were checked by HPLC and the appropriate fractionswere pooled to give 86 μmol of UTP labeled with Dye 108. The structureof the final product is given below:

Example 13 Preparation of dUTP Labeled with Dyes

For the preparation of labeled deoxyribonucleotides, the same proceduresdescribed in Examples 8, 10 and 12 were carried out except thatallylamine modified dUTP was substituted for the allylamine modifiedUTP.

Example 14 Synthesis of Dye 109 a) Preparation of Compound 14

A mixture of 15 g of 2-(methylthio)benzothiazole and 50 ml ofchlorosulfonic acid was heated to 60° C. for 2 hours. After cooling toroom temperature, the mixture was slowly added to 300 ml ice water withstirring. The white solid precipitate was collected by filtration andwashed with water to give 14 g of Compound 14 whose structure is givenbelow:

b) Preparation of Compound 15

To a solution of 13.2 g of Compound 14 from step (a) dissolved in 150 mlof dichloromethane, 15 ml of piperidine was slowly added and then thereaction mixture was stirred at room temperature overnight. The reactionmixture was shaken with 50 ml of 2N HCl in a separation funnel followedby the aqueous layer being discarded. The reaction mixture was thenshaken with 50 ml of saturated NaHCO₃ followed the aqueous layer beingdiscarded and the solution dried over MgSO₄. The organic solvent wasremoved by rotary evaporation and the solid residue Compound 15 wasdried in vacuum. The structure of Compound 15 is given below:

c) Preparation of Compound 16

The crude Compound 15 from step (b) was reacted with 50 mlp-Toluenesulfonic acid methyl ester at 140° C. for 2 hours. Aftercooling to room temperature, a precipitate formed which was collected byfiltration and washed with 50 ml of acetone and 150 ml of ether to give12 g of Compound 16 whose structure is given below:

d) Preparation of Compound 17

An intermediate compound, Compound 17, was prepared as described in U.S.Pat. No. 5,658,751 (herein incorporated by reference.). The structure ofthis compound is given below:

e) Preparation of Compound 18

A mixture of Compound 17 (2.8 g) from step d), POCl₃ (1.4 ml),dichloromethane (20 ml) and a catalytic amount DMF (5 μl) was refluxedovernight. The organic solvent was evaporated in vacuum, the residue(Compound 18) was washed with the mixture of ethyl acetate and ether(1:1, 2×50 ml). The structure of Compound 18 is given below:

f) Preparation of Compound 19

Compound 18 from step (e) was dissolved in 40 ml of dichloromethanefollowed by addition of 4 g of Compound 16 and 1.3 ml of triethylamine.The reaction mixture was stirred at room temperature for 8 hours. Thesolvent was removed by rotary evaporation and the residue was separatedby silica gel chromatography eluted with 10% methanol in methyl chlorideto give Compound 19 whose structure is given below:

g) Preparation of Dye 109

A mixture of Compound 19 from step f), N,N,N′-trimethylethylenediamineand 1,2-dichloroethane was heated at 55° C. for two hours. The solventwas evaporated and the residue was separated by silica gelchromatography eluted with 10% methanol in methyl chloride to give Dye109 whose structure is given below:

Example 15 Synthesis of Dye 110

Dye 109 from Example 14 was quarternized by refluxing Dye 109 withexcess methyl iodide overnight to give dye 110. The structure of Dye 110is given below:

Example 16 Synthesis of Dye 111

The preparation of Dye 111 was carried out as described in step (g) ofExample 14 except that instead of the N,N,N′-trimethylethylenediamineused in Example 14, N-(3-dimethylaminoethyl)-N-propylamine was mixedwith Compound 19 and 1,2-dichloroethane. The structure of Dye 111 is asfollows:

Example 17 Synthesis of Dye 112

Dye 111 from Example 16 was quarternized by refluxing Dye 111 withexcess methyl iodide overnight to give Dye 112. The structure of Dye 112is as follows:

Example 18 Synthesis of Dye 113 (a) Preparation ofN,N-dihexyl-2-methylbenzo[d]thiazole-6-sulfonamide (Compound 20)

A solution of compound Compound 2 (5.0 g, 20.2 mmol) from step (b) ofExample 1 dissolved in chloroform (30 ml) was added drop wise to asolution of dihexylamine (4.5 g, 24.2 mmol) in chloroform (20 ml). Thecombined mixture was stirred at room temperature for 2 hours. Thereaction mixture was then washed with water (2×, 50 ml) and brine (1×,50 ml). The organic layer was then dried with sodium sulfate andevaporated. The residue thus obtained was suspended in ca. 5 ml hotethyl acetate and this solution was then slowly added to 40 ml hexane.The white solid that precipitated was filtered and the filtrateevaporated to dryness to provide 7.76 g (97%) of Compound 20 as a syrupyliquid, R_(f)=0.43 (30% ethyl acetate in hexane). The structure of thiscompound is given below:

(b) Preparation of6-(N,N-dihexylsulfamoyl)-2,3-dimethylbenzo[d]-thiazole-3-ium tosylate(Compound 21)

A mixture of Compound 20 (2.0 g, 5.0 mmol) and p-toluenesulfonic acidmethyl ester (1.4 g, 7.5 mmol) was heated in a pressure tube at 130° C.for 2 hours. The mixture was allowed to cool to room temperature, andthe resulting mass was dissolved in ethyl acetate (4 ml). The combinedmixture was then added drop wise to hexane (40 ml). A sticky dark brownsolid separated which was washed with hexane and dried under vacuum toyield 2.3 g (78%) of Compound 21 which was used without any furtherpurification. The structure of this compound is given below:

(c) Preparation of 4-(dihexylamine) benzaldehyde (Compound 22)

This procedure was similar to the one described earlier in step (a) ofExample 1, except using N,N-di-N-hexylaniline (5.0 g, 19.1 mmol), POCl₃(4.5 g, 28.7 mmol) and DMF (55 ml). Compound 22 was obtained as a lightgreen liquid (5.06 g) and used without any further purification.R_(f)=0.15 (5% ethyl acetate in hexane). The structure of this compoundis given below:

(d) Preparation of Dye 113

A mixture of Compound 21 (0.50 g, 0.9 mmol), Compound 22 (0.30 g, 1.0mmol) and piperidine (40 μL, 0.4 mmol) was refluxed in ethanol (5 ml)for 18 hours. The reaction mixture was cooled to room temperature andmixed with 10 ml ethyl ether and the combined mixture was added to 100ml hexane. The precipitated solid was collected by centrifugation,washed with hexane (2×25 ml) and dried under vacuum to yield 0.26 g of apurple solid (Dye 113). Abs (max, methanol)=557 nm; Em=595 nm, ε=75,000M⁻¹cm⁻¹. The structure of Dye 113 is given below:

Example 19 Synthesis of Dye 114

A mixture of compound Compound 7 (0.17 g, 0.4 mmol) from step (a) ofExample 3, Compound 22 (0.13 g, 0.44 mmol) from step (c) of Example 18and piperidine (17 μL, 0.18 mmol) was refluxed in ethanol (2 ml) for 18hours. The reaction mixture was cooled to room temperature and mixedwith 30 ml ethyl ether. The precipitated solid was collected bycentrifugation, washed with ethyl ether (2×25 ml) and dried under vacuumto yield 0.23 g (83%) of a dark purple solid (Dye 114). Abs (max,methanol)=562 nm. The structure of Dye 114 is given below:

Example 20 Synthesis of Dye 115 (a) Preparation of Ethyl3-(4-methylquinolinium-1-yl) propylphosphonate (Compound 23)

A mixture of lepidine (1.0 g, 7.0 mmol) anddiethyl(3-bromopropyl)-phosphonate (2.0 g, 7.7 mmol) was heated in apressure tube at 130° C. for 4 hours. The mixture was allowed to cool toroom temperature, and the resulting mass was dissolved in DMF (4 ml).The combined mixture was then added drop wise to ethyl acetate (40 ml).An oily residue was obtained which was washed with ethyl acetate (2×40ml) and dried under vacuum to yield 1.9 g of Compound 23 which was thenused without any further purification. The structure of Compound 23 isgiven below:

(b) Preparation of Dye 115

A mixture of Compound 23 (0.53 g, 1.83 mmol), Compound 22 (0.67 g, 1.66mmol) from step (c) of Example 18 and piperidine (72 μL, 0.73 mmol) wasrefluxed in ethanol (5 ml) for 18 hours. The reaction mixture was cooledto room temperature and ethanol was evaporated to yield 0.9 g (70%) of adark blue solid (Dye 115). Abs (max, methanol)=570 nm; Em=640 nm,ε=27,000 M⁻¹cm⁻¹. The structure of Dye 115 is given below:

Example 21 Synthesis of Dye 116

This reaction was carried out as described previously in step (b) ofExample 3, using a mixture of Compound 23 (0.67 g, 1.66 mmol) from step(a) of Example 20, 9-ethyl-3-carbazolecarboxaldehyde (0.41 g, 1.83mmol), piperidine (72 μL, 0.73 mmol) and ethanol (5 ml). The resultantDye 116 was precipitated using ethyl ether, as a brown solid (0.78 g,yield: 68%). Abs (max, methanol)=494 nm. The structure of Dye 116 isgiven below:

Example 22 Synthesis of Dye 117

This process was carried out as described previously for Example 5except that in step (a), N,N′-diphenylformamidine (0.28 g, 1.4 mmol) wasmixed with Compound 7 (0.5 g, 1.2 mmol) from Example 3 instead ofCompound 4. This intermediate was then used as described previously instep (b) of Example 5 with1-(ε-carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate (0.72 g,2.0 mmol). The resultant Dye 117 was obtained as a purple solid (0.74g). The structure of Dye 117 is given below:

Example 23 Preparation of NHS Ester of Dye 117

This procedure was carried out as described previously in step (c) ofExample 5, using Dye 117 (0.7 g, 0.88 mmol) from Example 22,N-hydroxysuccinimide (0.51 g, 4.4 mmol), DCC (1.0 g, 5.3 mmol) and DMF(9 ml). The NHS ester of Dye 117 was obtained as a purple solid (0.53g). The structure of this compound is given below:

Example 24 Synthesis of Dye 118

A mixture of Compound 7 (1.0 g, 2.3 mmol) from step (a) of Example 3 andmalonaldehyde bis (phenylimine) monohydrochloride (0.8 g, 3.0 mmol) inacetic acid (5 ml) and acetic anhydride was heated at 120° C. for 2hours. The hot reaction mixture was then added to a solution of Compound12 from step (a) of Example 11, dissolved in a mixture of pyridine (15ml) and acetic anhydride (10 ml). The combined mixture was stirred atroom temperature for 18 hrs and then centrifuged to remove some coloredimpurities. The supernatant was then added drop wise to a 10-fold excessof ethyl acetate. The precipitated dark colored solid was collected bycentrifugation, washed with ethyl acetate and dried to yield 1.88 g ofDye 118. Abs (max, in 1× PBS pH 7.4)=651 nm; Em (max, in 1× PBS pH7.4)=667 nm. The structure of Dye 118 is given below:

Example 25 Preparation of NHS Ester of Dye 118

This procedure was carried out as described previously in step (c) ofExample 5, using Dye 118 (0.51 g, 0.62 mmol) from Example 24,N-hydroxysuccinimide (0.35 g, 3.0 mmol), DCC (0.62 g, 3.0 mmol) and DMF(10 ml). The NHS ester of Dye 117 was obtained as a dark blue solid (0.5g). The structure of this compound is given below:

Example 26 Synthesis of Dye 119

A mixture of Compound 23 (0.67 g, 1.66 mmol) from step (a) of Example20, 4-(dibutylamino)-benzaldehyde (0.43 g, 1.83 mmol) and piperidine (72μL, 0.73 mmol) was refluxed in ethanol (5 ml) for 18 hours. The reactionmixture was cooled to room temperature and the ethanol was evaporated.The residue thus obtained was subjected to silica gel chromatography andthe product eluted with 10% methanol in chloroform. Dye 119 was obtainedas a dark blue gummy paste (0.65 g, 70%). Abs (max, methanol)=565 nm;Em=637 nm. The structure of Dye 119 is given below:

Example 27 Synthesis of Dye 120 (a) Preparation ofN,N,2-trimethylbenzo[d]thiazole-6-sulfonamide (Compound 24)

A solution of Compound 2 (5.0 g, 20.2 mmol) from step (b) of Example 1dissolved in tetrahydrofuran (THF, 30 ml) was added drop wise to amixture of 2M N,N-dimethyl amine in THF (12 mL, 24.2 mmol) andtriethylamine (5.6 ml, 40.4 mmol). The resultant mixture was stirred atroom temperature for 3 hours. Solvents were removed by rotaryevaporation and the residue thus obtained was partitioned betweenchloroform (100 ml) and water (100 ml). The organic layer was thenwashed with water (2×, 100 ml) and brine (1×, 100 ml), dried over sodiumsulfate and evaporated. The oily residue thus obtained was dissolved inca. 5 ml of hot ethyl acetate and this solution was then slowly added to45 ml hexane. The mixture was cooled in the refrigerator overnight andthe white solid that precipitated was collected, washed with hexane anddried to yield 4.8 g (93%) of Compound 24 whose structure is givenbelow:

(b) Preparation of 6-(N,N-dimethylsulfamoyl)-2,3-dimethylbenzo[d]thiazole-3-ium tosylate (Compound 25)

A mixture of Compound 24 (2.0 g, 7.8 mmol) and p-toluenesulfonic acidmethyl ester (2.2 g, 11.7 mmol) was heated in a pressure tube at 130° C.for 2 hours. The mixture was allowed to cool to room temperature, andthe resulting mass was triturated with ethyl acetate (25 ml) until anoff-white solid separated. The solid was collected by centrifugation,washed with ethyl acetate and dried under vacuum to yield 2.9 g (84%) ofCompound 25 whose structure is given below:

(c) Preparation of Dye 120

A mixture of Compound 25 (1.0 g, 2.3 mmol) and malonaldehyde bis(phenylimine) monohydrochloride (0.66 g, 2.53 mmol) in acetic acid (5ml) and acetic anhydride was heated at 120° C. for 2 hours. The hotreaction mixture was then added to a solution of Compound 12 from step(a) of example 11 dissolved in pyridine (15 ml) and acetic anhydride (10ml). The resultant mixture was stirred at room temperature for 18 hoursand then centrifuged to remove some colored impurities. The supernatantwas then added drop wise to a 10-fold excess of ethyl acetate. The darkcolored precipitate was collected by centrifugation, washed with ethylacetate and dried to yield 1.86 g of Dye 120. Abs (max, in 1× PBS pH7.4)=652 nm. The structure of Dye 120 is given below:

Example 28 Preparation of NHS Ester of Dye 120

This procedure was carried out as described previously in step (c) ofExample 5, using Dye 120 (0.43 g, 0.64 mmol) from Example 27,N-hydroxysuccinimide (0.37 g, 3.2 mmol), DCC (0.66 g, 3.2 mmol) and DMF(10 ml). The NHS ester of Dye 120 was obtained as a dark blue solid (0.5g). The structure of this dye is given below:

Example 29 Synthesis of Dye 121 (a) Preparation of4-(6-(N.N-dimethylsulfamoyl)-2-methylbenzo[d]thiazole-3-ium-3-yl)butane-1-sulfonate(Compound 26)

A mixture of Compound 24 (2.55 g, 10.0 mmol) from step (a) of Example 27and 1-4-butane sultone (2.98 g, 21.9 mmol) was heated in a pressure tubeat 160° C. for 17 hours. The mixture was allowed to cool to roomtemperature, and the resulting mass was triturated with ethyl acetate(40 ml) until a pink solid separated. The solid was collected bycentrifugation, washed with ethyl acetate and dried under vacuum toyield 3.58 g (92%) of Compound 26 with the structure:

(b) Preparation of Compound 27

A mixture of Compound 12 (1.0 g, 2.12 mmol) from step (a) of Example 11and N, N′-diphenylformamidine (0.46 g, 2.33 mmol) in acetic acid (5 ml)was heated and refluxed for 1.5 hours. The reaction mixture was cooledand added to a mixture of ethyl ether (20 ml) and ethyl acetate (20 ml).The precipitated orange solid was collected by centrifugation, washedwith ethyl acetate (2×, 30 ml), dried under Argon and immediately usedin the next step.

(c) Preparation of Dye 121

Compound 27 was added to a suspension of Compound 26 (1.4 g, 3.6 mmol)dissolved in acetic anhydride (10 ml) and pyridine (10 ml). The combinedmixture was stirred in the dark at room temperature for 18 hours. Theprecipitated solid was collected by centrifugation, washed with apyridine/acetic anhydride mixture (1:1, 30 ml), washed with ethylacetate (2×, 30 ml) and dried to yield 0.71 g of a red solid (Dye 121).Abs (max, in water)=555 nm. The structure of Dye 121 is given below:

Example 30 Preparation of NHS Ester of Dye 121

This procedure was also carried out as described previously in step (c)of Example 5, using Dye 121 (0.25 g, 0.33 mmol) from Example 29,N-hydroxysuccinimide (0.19 g, 1.67 mmol), DCC (0.34 g, 1.67 mmol) andDMF (5 ml). The NHS ester of Dye 121 was obtained as a red solid (0.15g). The structure of this dye is given below:

Example 31 Fluorescence Enhancement of Various Dyes Upon Binding to aProtein

Some of the dyes of the preceding examples were tested for changes influorescence upon binding to a protein (BSA). 1 μM of each dye was mixedwith 150 μg of BSA in 10 mM TRIS (pH 7.5) with and without 0.05% SDS.Measurements were carried out on a standard fluorometer with and withoutBSA being present. Ratios were then calculated for the amount offluorescence (emission maxima) in the presence of BSA compared to itsabsence. These results are given in Table 1.

TABLE 1 Fluorescence Fluorescence Enhancement Enhancement without Dye #with SDS SDS 101 140 33 103 10 None 113 33 None 114 70 6 115 78 100 116None 12 119 92 95

Example 32 Preparation of Dye 122

Dye 109 from Example 14 was modified by dissolving in methanol andtreated with an excess of concentrated HCl at room temperature for 5minutes. After evaporation of solvents, the residue was washed withmethylene chloride/ethyl acetate (1:1) two times to give Dye 122 whosestructure is shown below:

Example 33 Amplification of Nucleic Acids in the presence of Dye 109

A target template was amplified on the Light Cycler (Roche) in thepresence of various amounts of Dye 109 from Example 14. As a control,various amounts of SYBR Green (Molecular Probes, Eugene, Oreg.) werealso included. The template was a 1:500 dilution of a 78 bp ampliconmade in a previous reaction from the anti-sense C construct described inLiu et al. 1997 (J. Virol. 71: 4079-4085). A 2×PCR mix was made up of 20mM Tris pH 8.3, 50 mM KCl, 4 mM MgCl₂, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mMdATP, 0.8 mM dUTP, 0.4 M Forward Primer and 0.4 mM Reverse Primer. Theprimers had the following sequences:

Forward Primer sequence = 5′ CAT GAT CCG GAT GGG AGG TG 3′ ReversePrimer sequence = 5′ GCA CAT CCG GAT AGT GAA TAG A 3′

A master mixture was made up of:

-   350 l of 2×PCR mix described above-   14 l of 25 mM MgCl₂-   122.5 l of DEPC treated H₂O-   14 ul of 1 u/l ung (Epicentre)-   7 l of 10 mg/ml BSA (New England Biolabs, Beverly, Mass.)-   7 l Taq polymease (Perkin Elmer)+7 l of TaqStart (Clontech)-   2.5 l of target template+2.0 l of various dilutions of dyes were    added to 15.5 l of Master mixture, incubated at 45° C. for 10    minutes and then amplified by:-   1) Melting    -   2 minutes @ 95° C.-   2) Cycling    -   a) Denaturation step: 0 seconds @ 95° C.    -   b) Annealing step: 5 seconds @ 69° C. for first cycle;        subsequent cycles were each reduced by 0.5° C. until 65° C. was        reached and all annealing steps in further cycles were carried        out at this temperature    -   c) Extension step: 5 seconds @ 72° C.-   3) After completion of 56 cycles, all samples were maintained at    4° C. until analyzed.

The samples were analyzed for detection of real time synthesis in theLight Cycler and it was seen that dye 109 could be used for this purpose(data not shown). After the amplification reactions were finished, thesamples were analyzed by gel electrophoresis. The gel analysis of thesereaction products is shown in FIG. 1. For SYBR Green, the recommendedreaction concentration is 1:10,000. As such, the amplification productsshown in lanes 5 and 6 using 2 l of 1:800 dilution in a 20 l reaction(final=1:8,000) is approximately representative of the product from thestandard procedure. For Dye 109, the reactions are listed in terms ofdilutions of a stock of Dye 109 with an OD reading of 200 taken at 495nm. As seen in FIG. 1, for both the 1:20 dilution of SYBR Green andundiluted Dye 109 stock, a strong inhibition of amplification was seenwith little or no product being synthesized. However, when lowerdilutions of the dyes were added there was no inhibition of the process.Furthermore, this Example demonstrates that at lower concentrations(1:200 dilution of SYBR Green in lane 7 and 1:25 dilution of Dye 109 inlanes 11 and 12), two unexpected but beneficial results were observed:a) enhancement of overall synthesis of target derived amplicons comparedto the reaction without any dye and b) elimination or suppression of thesynthesis of non-target derived amplicons seen in the absence of dye andat most of the tested dye concentrations.

Example 34 Amplification of Nucleic Acids in the Presence of Dye 109Using a Standard PCR Machine

The experiment shown in Example 33 was repeated using a standard PCRmachine for synthesis to see if the effects observed in Example 33 wasdependent upon the rapid heating and cooling steps used in the LightCycler.

A master mixture was made up of:

-   500 l of 2×PCR mix described in example 33-   20 l of 25 mM MgCl₂-   175 l of DEPC treated H₂O-   10 ul of 1 u/l ung (Epicentre)-   10 l of 10 mg/ml BSA (New England Biolabs, Beverly, Mass.)-   10 l Taq polymease (Perkin Elmer)+10 l of TaqStart (Clontech)

PCR conditions were 2.5 l of target template+2.0 l of dye was added to15.5 l of Master mixture incubated at 45° C. for 10 minutes and thenamplified using cycles of:

-   a) 94° C. for 30 seconds-   b) 65° C. for 30 seconds-   c) 72° C. for 1 minute

A duplicate set of samples was amplified with one set being taken outafter 20 cycles of amplification and the second set undergoing 40cycles. The gel analysis for this set of amplification reactions isshown in FIG. 2. It can be seen that after 20 cycles, a single discreteband can be seen for the 1:200 dilution of SYBR Green (Lanes 5 and 6)whereas all other SYBR Green dilutions and in the absence of any dye, nosynthesis was evident. Similarly for Dye 109, a single discrete band canbe seen for the 1:10 dilution (lanes 12 and 13), lesser amounts ofsynthesis with the 1:15 dye dilution samples (lanes 14 and 15) and faintbands at slightly lower dye concentrations (1:20 and 1:25 in lanes 16/17and 18/19 respectively). This indicates that under these conditionsthere was more efficient synthesis of amplicons in the presence of SYBRGreen and Dye 109 at these concentrations, qualitatively repeating theresults of Example 33. After 40 cycles, synthesis was observed at allconcentrations except the high dye samples in lanes 7, 9, 10 and 11. Theother effect previously observed was also seen, i.e. non-target ampliconsynthesis seen in the no Dye reaction (lane 1) was reduced with the1:200 dilution of SYBR Green and the 1:10 dilution of Dye 109 againrepeating the results of Example 33.

Example 35 Amplification of Nucleic Acids in the Presence of Dye 122 and109a

A target template was amplified on the Light Cycler (Roche) in thepresence of various amounts of SYBR Green and Dye 122 from Example 32.In addition, a byproduct from the silica gel chromatography of Dye 109in step (g) of Example 14 was also used. This compound is believed tohave a single protonation of one of the amines of Dye 109 and as suchwill be termed Dye 109a. The stock of Dye 122 was OD=200 measured at 488nm and the stock of Dye 109a was OD=200 measured at 498 nm. The templatewas a 1:50,000 dilution of the 78 bp amplicon of the anti-sense Cconstruct described previously.

A master mixture was made up of:

-   350 l of 2×PCR mix described in Example 33-   14 l of 25 mM MgCl₂-   175.0 l of DEPC treated H₂O-   7 l of 10 mg/ml BSA (New England Biolabs, Beverly, Mass.)-   7 l Taq polymease (Perkin Elmer)+7 l of TaqStart (Clontech)

2.0 l of target template+2.0 l of various dye dilutions were added to 16l of Master mixture, and then amplified as described previously inExample 33 except that the incubation at 45° C. for 10 minutes waseliminated since ung was not used for this series of amplifications. Theamplification was stopped after 19 cycles. The results of this series ofamplifications are seen in FIG. 3. For SYBR Green, the 1:800 dilution(lanes 4 and 5) gave the best amplification with a sharp drop off oneither side. For Dye 122, the 1:10 dilution (lane 10) was inhibitorywhile the 1:30 dilution (lanes 11 and 12) was the best with lessamplification seen for the next dye dilutions (1:100 etc.). For Dye109a, similar features were seen where 1:10 was inhibitory (lane 18) and1:30 was optimal (lanes 19 and 20).

Example 36 Amplification of Nucleic Acids in the Presence of Dye 122 and109a

Although the previous example showed a significant improvement ofamplification for Dye 109a and 122, there was a fairly substantialdifference between the best concentration of dye and the next higher andlower dilutions of dyes. A series of dilutions that were closer togetherwas carried out for both Dye 109a and Dye 122. The target template wasamplified on the Light Cycler and the template was the 1:50,000 dilutionof the 78 bp amplicon of the anti-sense C construct describedpreviously.

A master mixture was made up as described previously in Example 35.

2.0 l of target template (1:50,000 dilution of the antisense Camplicon)+2.0 l of various dye concentrations were added to 16 l ofMaster mix and then amplified as described previously in Example 33 andstopping after 17 amplification cycles. Samples taken from theseamplification reactions are shown in FIG. 4. As seen in these results, afairly broad spectrum of dilutions can give a strong enhancement effectfor both Dye 122 and Dye 109a.

Example 37 Amplification of Various Amounts of Nucleic Acids in thePresence of Dye 122 and 109a

In the previous experiments, a fixed amount of target was amplified inthe presence of varying level of dyes. The eventual use of these dyes isgoing to be with a fixed amount of dye and varying levels of targets. Assuch, an experiment was carried out using previously determined optimallevels of dyes and various amounts of input target (again using theanti-sense C as a model).

-   Dilution 1=1:100,000-   Dilution 2=1:1,000,000-   Dilution 3=1:10,000,000-   Dilution 4=1:100,000,000-   Dilution 5=1:1,000,000,000-   Dilution 6=1:10,000,000,000

Selected amounts of target template were amplified on the Light Cycler(Roche) in the presence of various amounts of Dye 122, Dye 109a and SYBRGreen.

A master mixture was made up of:

-   380 l of 2×PCR mix described in Example 33-   15.2 l of 25 mM MgCl₂-   190.0 l of DEPC treated H₂O-   7.6 l of 10 mg/ml BSA (New England Biolabs, Beverly, Mass.)-   7.6 l Taq polymease (Perkin Elmer)+7.6 l of TaqStart (Clontech)

2.0 l of target template (1:50,000 dilution of the antisense Camplicon)+2.0 l of various dye concentrations were added to 16 l ofMaster mixture and amplified as described previously in Example 33stopping after 29 cycles. Dye additions were from the 1:1000 dilution ofSYBR Green, 1:30 of Dye 109a and 1:30 of Dye 122. Samples taken fromthese amplification reactions are shown in FIG. 5.

At the highest input level (Dil 2 1:1,000,000 dilution of amplicon) noevidence of amplification is seen in the sample without dye. On theother hand, a faint but visible band can be seen for the sample thatincluded SYBR Green. Further dilutions of target do not show anyevidence of an amplification product for the SYBR Green samples. Moreinterestedly, it can be seen that for the samples that included Dyes109a and 122, a strong prominent band can be seen with the Dil 2(1:1,000,000) input level and visible bands for the inputs that had beendiluted 10-100× more.

Many obvious variations will be suggested to those of ordinary skill inthe art in light of the above detailed descriptions of the presentinvention. All such obvious variations are fully contemplated and areembraced by the scope and spirit of the present invention as set forthin the claims that now follow.

1. A method for detecting the presence or quantity of a targetcomprising the steps of: a) providing i) a sample where the presence orquantity of a target is desired to be detected, and ii) a compositioncomprising a first portion and a second portion wherein said firstportion comprises a dye and said second portion comprises a targetspecific moiety; b) allowing any targets present in said sample to bindwith said target specific moiety; and c) detecting or quantifying theamount of said dye bound to any of said target in the sample, therebydetecting the presence or quantity of said target, wherein the dyecomprises


2. The method of claim 1, wherein said target comprises anoligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide,enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor,lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin,carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viralparticle, viral component, bacterial cell, bacterial component,eukaryotic cell, eukaryotic cell component, natural drug, syntheticdrug, or organic molecule.
 3. The method of claim 1, wherein said dyefurther comprises one or more charged groups or polar groups.
 4. Themethod of claim 3, wherein at least one of said one or more chargedgroups or polar groups is monomeric.
 5. The method of claim 3, whereinat least one of said one or more charged groups or polar groups ispolymeric.
 6. The method of claim 3, wherein at least one of said one ormore charged groups or polar groups is anionic.
 7. The method of claim3, wherein at least one of said one or more charged groups or polargroups is cationic.
 8. The method of claim 1, wherein said dye furthercomprises one or more anionic charged groups or polar groups and one ormore cationic charged groups or polar groups.
 9. The method of claim 1,wherein said target specific moiety comprises an oligonucleotide,polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen,antibody, hormone, hormone receptor, cellular receptor, lymphokine,cytokine, hapten, lectin, avidin, streptavidin, digoxigenin,carbohydrate, oligosaccharide, polysaccharide, natural drug, syntheticdrug, or organic molecule.
 10. The method of claim 1, wherein said dyeis linked to said target specific moiety through a linker arm.
 11. Themethod of claim 10, wherein said linker arm is attached to said targetspecific moiety through a bond which comprises a covalent bond, a polarbond or a coordinate bond.
 12. The method of claim 9, wherein saidtarget specific moiety is an oligonucleotide or polynucleotide thatcomprises two or more dye molecules attached to separate nucleotides ofsaid oligonucleotide or polynucleotide.
 13. The method of claim 12,wherein said two or more dye molecules comprise the same dye molecules.14. The method of claim 12, wherein said two or more dye moleculescomprise different dye molecules.
 15. The method of claim 1, whereinsaid dye is part of a composite dye comprising a second dye molecule.16. The method of claim 9, wherein said target specific moiety is anoligonucleotide or polynucleotide that comprises unmodified nucleotides,modified nucleotides, nucleotide analogues or any combination thereof.17. The method of claim 1, wherein said sample i) or said compositionii) is fixed or immobilized to a solid support.
 18. The method of claim17, further comprising carrying out one or more washing steps after saidbinding step b).
 19. The method of claim 1, wherein said binding step b)and said detecting step c) are carried out in the absence of washingsteps.
 20. The method of claim 19, wherein said detection step c) iscarried out by energy transfer.
 21. A method for detecting the presence,location or quantity of a target comprising the steps of: a) providingi) a sample where the presence, location or quantity of a target isdesired to be detected, and ii) a dye; b) allowing any targets presentin said sample i) to bind with said dye ii); and c) detecting the dyeii) bound to any of said target in the sample i), thereby detecting thepresence, location or quantity of said target, wherein the dye comprises


22. The method of claim 21, wherein said target comprises anoligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide,enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor,lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin,carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viralparticle, viral component, bacterial cell, bacterial component,eukaryotic cell, eukaryotic cell component, natural drug, syntheticdrug, or organic molecule.
 23. The method of claim 21, wherein saidtarget has been bound to a solid matrix.
 24. The method of claim 23,wherein said solid matrix comprises a bead, filter, gel, cuvette, testtube, dish, microscope slide, plate, microtiter plate or microarray. 25.The method of claim 24, wherein said target is detected in situ.
 26. Themethod of claim 21, wherein said detection takes place without bindingof target to a solid matrix.
 27. The method of claim 26, wherein saiddetection takes place by a change in properties of said dye afterbinding takes place between said dye and said target.
 28. The method ofclaim 27, wherein said change is an increase or decrease in the amountof fluorescence of said dye.
 29. The method of claim 23, wherein afurther step is carried out where unbound dye is removed.
 30. The methodof claim 1 or 21, wherein the dye comprises


31. The method of claim 1 or 21, wherein the dye comprises


32. The method of claim 1 or 21, wherein the dye comprises


33. The method of claim 1 or 21, wherein the dye comprises


34. The method of claim 1 or 21, wherein the dye comprises


35. The method of claim 1 or 21, wherein the dye comprises


36. The method of claim 1 or 21, wherein the dye comprises


37. The method of claim 1 or 21, wherein the dye comprises


38. The method of claim 1 or 21, wherein the dye comprises


39. The method of claim 1 or 21, wherein the dye comprises


40. The method of claim 1 or 21, wherein the dye comprises


41. The method of claim 1 or 21, wherein the dye comprises


42. The method of claim 1 or 21, wherein the dye comprises


43. The method of claim 1 or 21, wherein the dye comprises


44. The method of claim 1 or 21, wherein the dye comprises


45. The method of claim 1 or 21, wherein the dye comprises


46. The method of claim 1 or 21, wherein the dye comprises


47. The method of claim 1 or 21, wherein the dye comprises


48. The method of claim 1 or 21, wherein the dye comprises


49. The method of claim 1 or 21, wherein the dye comprises


50. The method of claim 1 or 21, wherein the dye comprises


51. The method of claim 1 or 21, wherein the dye comprises


52. The method of claim 1 or 21, wherein the dye comprises